Use of MVA to treat prostate cancer

ABSTRACT

The invention relates to compositions, kits, and methods for cancer prophylaxis and therapy using recombinant MVA viruses encoding tumor-associated antigens, such as PSA and PAP. The recombinant MVA viruses can induce B- and T-cell responses. The recombinant MVA viruses can be administered prior to, at the same time as, or after a taxane.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional Application No.60/960,893, filed Oct. 18, 2007.

FIELD OF THE INVENTION

The invention relates to the prophylaxis and treatment of cancers,particularly prostate cancer, using MVA viruses encodingtumor-associated antigens, particularly prostate-specific antigen (PSA)and prostatic acid phosphatase (PAP).

BACKGROUND OF THE INVENTION

Modified Vaccinia Ankara (MVA) virus is related to vaccinia virus, amember of the genera Orthopoxvirus, in the family of Poxviridae. MVA wasgenerated by 516 serial passages on chicken embryo fibroblasts of theAnkara strain of vaccinia virus (CVA) (for review see Mayr, A., et al.Infection 3:6-14 (1975)). As a consequence of these long-term passages,the genome of the resulting MVA virus had about 31 kilobases of itsgenomic sequence deleted and, therefore, was described as highly hostcell restricted for replication to avian cells (Meyer, H. et al., J.Gen. Virol. 72:1031-1038 (1991)). It was shown in a variety of animalmodels that the resulting MVA was significantly avirulent (Mayr, A. &Danner, K., Dev. Biol. Stand. 41:225-34 (1978)). Additionally, this MVAstrain has been tested in clinical trials as a vaccine to immunizeagainst the human smallpox disease (Mayr et al., Zbl. Bakt. Hyg. I, Abt.Org. B 167:375-390 (1987); Stickl et al., Dtsch. med. Wschr.99:2386-2392 (1974)). These studies involved over 120,000 humans,including high-risk patients, and proved that, compared tovaccinia-based vaccines, MVA had diminished virulence or infectiousness,while it induced a good specific immune response. In the followingdecades, MVA was engineered for use as a viral vector for recombinantgene expression or as a recombinant vaccine (Sutter, G. et al., Vaccine12:1032-40 (1994)).

Even though Mayr et al. demonstrated during the 1970s that MVA is highlyattenuated and avirulent in humans and mammals, certain investigatorshave reported that MVA is not fully attenuated in mammalian and humancell lines since residual replication might occur in these cells.(Blanchard et al., J Gen Virol 79:1159-1167 (1998); Carroll & Moss,Virology 238:198-211 (1997); Altenberger, U.S. Pat. No. 5,185,146;Ambrosini et al., J Neurosci Res 55(5):569 (1999)). It is assumed thatthe results reported in these publications have been obtained withvarious known strains of MVA, since the viruses used essentially differin their properties, particularly in their growth behavior in variouscell lines. Such residual replication is undesirable for variousreasons, including safety concerns in connection with use in humans.

Strains of MVA having enhanced safety profiles for the development ofsafer products, such as vaccines or pharmaceuticals, have beendescribed. See U.S. Pat. Nos. 6,761,893 and 6,193,752. Such strains arecapable of reproductive replication in non-human cells and cell lines,especially in chicken embryo fibroblasts (CEF), but are not capable ofreproductive replication in certain human cell lines known to permitreplication with known vaccinia strains. Those cell lines include ahuman keratinocyte cell line, HaCat (Boukamp et al. J Cell Biol106(3):761-71 (1988)), a human cervix adenocarcinoma cell line, HeLa(ATCC No. CCL-2), a human embryo kidney cell line, 293 (ECACC No.85120602), and a human bone osteosarcoma cell line, 143B (ECACC No.91112502). Such viral strains are also not capable of reproductivereplication in vivo, for example, in certain mouse strains, such as thetransgenic mouse model AGR 129, which is severely immune-compromised andhighly susceptible to a replicating virus. See U.S. Pat. No. 6,761,893.One such MVA strain and its derivatives and recombinants, referred to as“MVA-BN®,” have been described. See U.S. Pat. Nos. 6,761,893 and6,193,752.

MVA and MVA-BN® have each been engineered for use as a viral vector forrecombinant gene expression or as a recombinant vaccine. See, e.g.,Sutter, G. et al., Vaccine 12:1032-40 (1994), U.S. Pat. Nos. 6,761,893and 6,193,752.

Cancer-related diseases are a leading cause of mortality and morbidityworldwide. For example, in the US alone, it is estimated that one in sixmen will suffer from prostate cancer. Moreover, autopsy studies showthat a significant proportion of the male population is known to carrythe disease, albeit at its earliest non-malignant stages, as early as bythe age of 30. See, e.g., Taichman et al., JCI 117(9):2351-2361 (2007);Webster et al., J. Clin. Oncol. 23:8262-8269 (2005). Recent approachesto cancer immunotherapy have included vaccination with tumor-associatedantigens. In certain instances, such approaches have included use of adelivery system to promote host immune responses to tumor-associatedantigens. Such delivery systems have included recombinant viral vectors,as well as cell-based therapies. See, e.g., Harrop et al., Front.Biosci. 11:804-817 (2006); Arlen et al., Semin. Oncol. 32:549-555(2005); Liu et al., Proc. Natl. Acad. Sci. USA 101 (suppl.2):14567-14571 (2004). MVA has been used as a vaccine vehicle for the5T4 oncofetal antigen in clinical trials in metastatic colorectal,metastatic renal and hormone-refractory prostate cancer patients. Amato,R J., Expert Opin. Biol. Ther. 7(9): 1463-1469 (2007).

Among the known tumor-associated antigens are prostate-specific antigen(PSA) and prostatic acid phosphatase (PAP). See, e.g., Taichman et al.,JCI 117(9): 2351-2361 (2007); Webster et al., J. Clin. Oncol.23:8262-8269 (2005). PSA is produced by the prostate and is found in anincreased amount in the blood of men who have prostate cancer, benignprostatic hyperplasia, or infection or inflammation of the prostate. PSAhas been identified as a target for cell-mediated immunotherapyapproaches to cancer treatment. See, e.g., McNeel, D. G., Curr. Opin.Urol. 17:175-181 (2007); Nelson W. G., Curr. Opin. Urol. 17:157-167(2007). PAP is an enzyme measured in the blood whose levels may beelevated in patients with prostate cancer which has invaded ormetastasized elsewhere. PAP is not elevated unless the tumor has spreadoutside the anatomic prostatic capsule, either through localizedinvasion or distant metastasis. Therefore this prostate tumor antigen isbeing investigated as a target antigen in several human vaccine trials,some with evidence of clinical benefit. See, e.g., McNeel, D. G., Curr.Opin. Urol. 17:175-181 (2007); Waeckerle-Men et al., Cancer Immunol.Immunother. 66:811-821 (2006); Machlenkin et al. Cancer ImmunolImmunother. 56(2):217-226 (2007).

PAP containing vaccines have been generated using recombinant vacciniavirus, purified PAP, DNA vaccines, and antigen-loaded dendritic cells.Valone et al., The Cancer Journal 7: Suppl 2:S53-61 (2001); Fong et al.,J Immunol. 2001 Dec. 15;167(12):7150-6; Fong et al., J. Immunol.159(7):3113-7 (1997); Johnson et al., Vaccine 24(3):293-303 (2006);Johnson et al., Cancer Immunol Immunother. 56(6):885-95 (2007). In onestudy, no antibodies to PAP were detected when dendritic cells pulsedwith PAP-GM-CSF were injected into rats. (Valone et al. at S55.). Inanother study, administration of recombinant vaccinia virus containinggenes encoding rat PAP or human PAP did not generate a measurableantibody response to rat or human PAP. (Fong et al. (1997) at 3116-7.)In another study, PAP-specific IgG could be detected in the sera ofanimals immunized with hPAP protein as well as in animals that receivedvaccinia virus encoding human PAP vaccination followed by hPAP proteinas a booster immunization, but not in animals immunized twice withvaccinia virus encoding human PAP. (Johnson et al. (2007) at 890.)

Active cancer immunotherapy relies on the induction of an immuneresponse against tumor cells in cancer patients. The induction of bothhumoral and cellular components of adaptive immunity against a broadrange of tumor-associated antigens (TAA) and the concomitant activationof components of innate immunity are essential for maximal efficacy ofan active immunotherapy product. Specifically, Type 1 or Th1 adaptiveimmunity characterized by the induction of antigen-specificIFNγ-producing cytotoxic T-cells (CD8 T-cells) is believed to beimportant for anti-cancer immunotherapy.

Despite the recent advances in cancer treatment, prostate cancer remainsthe second leading cause of death among American cancer patients. Thus,therapeutic approaches that might better alleviate the disease bytargeting multiple aspects of tumor growth and metastasis are needed.

Taxanes, such as paclitaxel and docetaxel, have been used aschemotherapies for cancer patients. See, e.g., Tannock et al., N. Engl.J. Med. 351:1502-1512 (2004). Chemotherapy with taxanes has beencombined with different tumor vaccine treatments, resulting in a varietyof results. See, Chu et al., J. Immunotherapy 29:367-380 (2006);Machiels et al., Cancer Res. 61:3689-3697 (2001); Prell et al., CancerImmunol. Immunother. 55:1285-1293 (2006); Arlen et al., Clinical BreastCancer 7:176-179 (2006); and Arlen et al., Clinical Cancer Res.12:1260-1269 (2006). The combination of cancer vaccines withchemotherapies has been reviewed in Chong et al., Expert Opin.Phamacother. 6:1-8 (2005); Emens et al., Endocrine-Related Cancer12:1-17 (2005); and McNeel, D. G., Curr. Opin. Urol. 17:175-181 (2007).

Based on the above, a need in the art exists for reagents and methodsfor cancer therapy.

BRIEF SUMMARY OF THE INVENTION

The invention encompasses methods, reagents, and kits for cancerprophylaxis and the treatment of cancer patients, both of primary tumorsand also of cancer metastasis.

The invention encompasses a method for treating a human cancer patientcomprising administering to the patient a recombinant MVA encoding apolypeptide comprising a human prostate-specific antigen (PSA) antigenand a polypeptide comprising a human prostatic acid phosphatase (PAP)antigen. In one embodiment, the MVA is MVA-BN. In one embodiment, theMVA virus comprises the nucleotide sequence of SEQ ID NO:1 and SEQ IDNO:2. In one embodiment, the PSA antigen and the PAP antigen areinserted in the MVA intergenic region 014L/015L. In certain embodiments,the cancer is prostate cancer or a prostate cancer metastasis.

In one embodiment, the recombinant MVA is administered prior to atumoricidal dose of a taxane. In one embodiment, the recombinant MVA isadministered at the same time as a tumoricidal dose of a taxane. In oneembodiment, the recombinant MVA is administered after a tumoricidal doseof a taxane. In preferred embodiments, the taxane is docetaxel orpaclitaxel.

The invention encompasses kits for the prophylaxis of prostate cancercomprising a recombinant MVA encoding a polypeptide comprising a humanPSA antigen and a polypeptide comprising a human PAP antigen andinstructions to administer the recombinant MVA prior to the detection ofprostate cancer.

The invention encompasses kits for the treatment of prostate cancercomprising a recombinant MVA encoding a polypeptide comprising a humanPSA antigen and a polypeptide comprising a human PAP antigen andinstructions to administer the recombinant MVA to a prostate cancerpatient.

The invention encompasses kits for treating a cancer patient comprisinga recombinant MVA encoding a polypeptide comprising a human PSA antigenand a polypeptide comprising a human PAP antigen and instructions toadminister the recombinant MVA prior to, at the same time as, or aftertreatment with a tumoricidal dose of a taxane.

The invention encompasses a recombinant MVA virus expressing apolypeptide comprising a human PAP antigen. In one embodiment, the MVAvirus comprises SEQ ID NO:2. In one embodiment, the MVA is MVA-BN.

The invention encompasses a recombinant MVA virus expressing apolypeptide comprising a human PSA antigen and a polypeptide comprisinga human PAP antigen. In one embodiment, the MVA virus comprises thenucleotide sequence of SEQ ID NO:1. In one embodiment, the MVA viruscomprises the nucleotide sequence of SEQ ID NO:2. In one embodiment, theMVA is MVA-BN.

The invention encompasses an immunogenic composition comprising arecombinant MVA virus encoding a polypeptide comprising a human PAPantigen, wherein the immunogenic composition induces B-cell and T-cellimmune responses against PAP when administered to a host.

The invention encompasses an immunogenic composition comprising arecombinant MVA virus encoding a polypeptide comprising a human PAPantigen, wherein the immunogenic composition induces antibodies againstPAP when administered to a host. In one embodiment, the MVA viruscomprises SEQ ID NO:2.

The invention encompasses an immunogenic composition comprising arecombinant MVA virus encoding a polypeptide comprising a human PSAantigen and a polypeptide comprising a human PAP antigen, wherein theimmunogenic composition induces B-cell and T-cell immune responsesagainst PSA and PAP when administered to a host.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. Schematic maps of MVA-BN® genome. Shown in 1A are thelocations of the six deletion sites in the MVA-BN® genome: shadedsections and letters (A to O) identify HindIII restriction enzymedigestion fragments, and the positions and sizes of the CVA sequencesthat are lacking in the MVA-BN® are shown on the arrows. Shown in 1B arethe HindIII restriction fragments (letters A to O) and the IGR 014L/015Lsite used to generation of MVA-BN-PRO.

FIG. 2. Schematic Overview of the MVA-BN-PRO virus. Map of the MVA-BN®genome (HindIII restriction map, indicated by letters A to O) outliningthe recombinant insert cloned in the intergenic region 014L/015L: PSAand PAP genes, each under the control of the cowpox virus ATI promoter(ATI).

FIG. 3A-B. PAP (A) and PSA (B) detection in supernatant of CT26 cellcultures incubated with MVA-BN-PRO. CT26 cells at a density of 6×10⁵cells per well (dark squares and triangles) or 6E4 cells per well (greysquares) were infected with either MVA-BN-PRO (squares) or MVA-BN®(triangles) at indicated multiplicity of infection (MOI). 24 hourslater, cell supernatants were harvested and PAP and PSA protein levelswere measured by PAP enzymatic assay and PSA ELISA, respectively.

FIG. 4A-B. Anti-PSA (A) and Anti-PAP (B) Antibody Responses in MiceTreated with MVA-BN-PRO. Animals were immunized three times (day 1, 15,and 29) with either MVA-BN-PRO (white squares) or MVA-BN® (blacksquares). Blood samples were collected before treatment, at day 14, 28,and 42. Titers are the reciprocal value of the last dilution with anO.D. at least 2-fold higher than the background (serum at same dilutionfrom TBS treated animals). Titers indicated as zero were negative at thelowest serum dilution tested (1:50).

FIG. 5A-B. Anti-PSA and Anti-PAP T-cell Responses in Mice Treated withMVA-BN-PRO. Splenocytes from MVA-BN-PRO immunized animals (A) and TBScontrol animals (B) were incubated with OPL from PAP (gray squares), PSA(white squares) or HER2 (black squares) sequences at the indicatedconcentrations. Spots indicative of IFN-γ-producing T-cells werenumerated using an ImmunoSpot Analyzer. Means from triplicate wells andstandard deviation are represented for each OPL concentration tested.

FIG. 6A-B. CD4 and CD8 T-cell Contributions to MVA-BN-PRO MediatedT-cell Responses. Animals were immunized with MVA-BN-PRO four times (d1, 15, 29, and 49) and splenocytes were collected six days after thelast treatment, CD8 depleted splenocytes (A) and CD4 depletedsplenocytes (B) were incubated with OPL from PAP (gray squares), PSA(white squares) or HER2 (black squares) sequences at the indicatedconcentrations. The analysis was carried out as described for FIG. 5.

FIG. 7A-F. Prophylatic prevention of tumor growth in mice treated withMVA-BN-PRO. Animals were treated 3 times at 3 week intervals atindicated TCID50 of virus diluted in TBS. Six weeks after the thirdtreatment animals were challenged intradermally with 1×10⁵PSA-expressing E5 tumor cells. Tumor growth was measured twice weeklyusing calipers. Tumor volume was calculated as: (L×W²)/2. 7A through 7E:tumor growth in individual mice is reported for each treatment group.7F: mean tumor sizes and standard deviation are reported for alltreatment groups.

FIG. 8. Prevention of Tumor Growth in Mice Treated with MVA-BN-PRO.Comparison of Day 29 Measurements in two Separate Experiments. Animalswere treated three times at 2-week intervals (grey symbols) with 2×10⁶TCID₅₀ of indicated virus diluted in TBS. Two weeks after the lasttreatment, animals were challenged intradermally with 1×10⁵PSA-expressing E5 tumor cells. Tumor growth was measured twice weeklyusing calipers. Tumor volume was calculated as: (L×W²)/2. Dots showtumor volumes for each animal on Day 29 post tumor implantation. Datafrom the matching groups of a separate experiment described in FIG. 7are represented in black symbols for comparison. Both experimentsreported here were conducted under similar conditions except for thelength of treatment intervals (3-week vs 2-week intervals) and the timeof tumor cell implantation (six vs. two weeks after the thirdtreatment).

FIG. 9A-F. Therapeutic Suppression of Tumor Growth in Mice Treated withMVA-BN-PRO. BALB/c mice (10 animals in each group) were challenged withE6 cells (1×10⁵ cells injected id) on day 1 and treated subcutaneouslyon day 1, 8, and 15 either with TBS (E), MVA-BN® (5×10⁶ or 5×10⁷ TCID₅₀;A and B), or MVA-BN-PRO (5×10⁶ or 5×10⁷ TCID₅₀; C and D). Mice weresacrificed on day 22. Panels A-E show tumor sizes of individual mice.Averages of tumor sizes and standard deviations for each group aredepicted in panel F. Tumor growth was measured twice weekly usingcalipers. Tumor volume was calculated as: (L×W²)/2. Error bars representstandard deviations (SD).

FIG. 10. Suppression of PAP-positive Tumor Growth in Mice Treated withMVA-BN-PRO. BALB/c mice (10 animals in each group) were challenged withCT26-PAP (5×10⁵ cells injected intravenously) on Day 1 and treatedintraperitonally on Day 4 either with TBS, MVA-BN (5×10⁷ TCID₅₀), orMVA-BN-PRO (2×10⁶ and 5×10⁷ TCID₅₀). Mice were sacrificed on Day 14 andtheir lungs weighed. Data points represent the lung weight of individualmice. Horizontal bar indicate the mean of lung weight for each group.

FIG. 11. Anti-PSA and anti-PAP antibody responses induced in BALB/c orC57BL/6 mice. Male BALB/c and C57BL/6 mice (5 animals in each group)were immunized on days 1, 15, and 29 with 5×10⁷ TCID₅₀ of MVA-BN-PRO.Blood samples were collected on day 42, and serial dilutions of pooledsera were analyzed for the presence of anti-PSA or anti-PAP IgG byELISA. Titers were calculated as the reciprocal value of the lastdilution with an O.D. at least 2-fold higher than background (defined asserum at the same dilution from TBS treated animals). Data points forsera with titers below the lowest dilution tested (<125) werearbitrarily placed on the x-axis positioned one dilution below the firstdilution of the assay (62.5) for graphing purposes.

FIG. 12. T cell responses in Patient treated with MVA-BN-PRO. PBMC fromblood of Patient J-D-1001 were collected pre-treatment (Base) orpost-MVA-BN-PRO treatment (TC3). Cells were incubated for 40 hours witheither PSA protein, PSA overlapping peptide library (OPL), PAP protein,PAP OPL, pools of MHC Class I and Class II peptides derived from tumorassociated antigens (TAA) or MVA-BN at the concentration indicated onthe graph. T cell activation was determined by ELISpot measuringsecreted IFN-γ. For each stimulating condition, results are expressed asthe mean IFN-γ spot forming cells (SFC) per 2×10⁵ PBMC. SFC values werederived from the mean of quadruplicate wells with the backgroundsubtracted.

DETAILED DESCRIPTION OF THE INVENTION

A recombinant MVA expressing human PSA and PAP antigens (MVA-BN-PRO) wastested in a panel of in vitro and in vivo assays. The expression of bothprostate-specific antigens encoded by MVA-BN-PRO (PSA and PAP) ineukaryotic cells incubated with MVA-BN-PRO was evaluated using a PSAdetection kit and a functional assay for phosphatase activity,respectively. ELISA and ELISpot assays were used to monitor theinduction of anti-PSA and anti-PAP antibody and T-cell immune responsesin mice treated with MVA-BN-PRO. The anti-tumor activity of MVA-BN-PROwas assessed in PSA-tumor models, both in a prophylactic setting and ina therapeutic setting.

These studies demonstrated that (i) uptake of MVA-BN-PRO by cells invitro results in expression of both PAP and PSA in similar amounts; (ii)treatment of mice with MVA-BN-PRO results in anti-PSA and anti-PAPhumoral and Th1 cellular immune responses, (iii) treatment of mice withMVA-BN-PRO inhibits the growth of PSA (+) tumors in both prophylacticand therapeutic settings, (iv) treatment of mice with MVA-BN-PROinhibits the growth of PAP (+) tumors in a therapeutic setting, (v) in ahuman, MVA-BN-PRO treatment increased levels of both anti-PSA T cellsand anti-PAP T cells, and (vi) MVA-BN-PRO treatment in a human resultedin the spreading of T cell responses to other tumor antigens. Thus,MVA-BN-PRO activates the immune system by triggering antigen-specifichumoral and cellular Th1-type responses, which results in significanttherapeutic activity against PSA and PAP expressing tumors in vivo.Consequently, MVA-BN-PRO is an attractive vaccine candidate for theimmunotherapy of prostate cancer in humans.

MVA-BN-PRO is a potent immunogen able to induce protective anti-tumorimmunity that prevents tumor growth in a prophylactic setting and thatalso suppresses the growth of established tumors. The prophylactic andtherapeutic anti-tumor activities of MVA-BN-PRO were mediated byanti-PSA-specific adaptive immune responses. However, adaptive immuneresponses were induced against both prostate-specific antigens, PSA andPAP, encoded by MVA-BN-PRO. The concomitant activation of adaptiveresponses against multiple tumor antigens enables MVA-BN-PRO to combattumors more efficiently and increase the potential to successfully treatcancer patients.

In one embodiment, the invention encompasses the use of recombinant MVAviruses for prostate cancer therapy. The recombinant MVAs are generatedby insertion of heterologous sequences into an MVA virus. Examples ofMVA virus strains that are useful in the practice of the presentinvention and that have been deposited in compliance with therequirements of the Budapest Treaty are strains MVA 572, deposited atthe European Collection of Animal Cell Cultures (ECACC), VaccineResearch and Production Laboratory, Public Health Laboratory Service,Centre for Applied Microbiology and Research, Porton Down, Salisbury,Wiltshire SP4 OJG, United Kingdom, with the deposition number ECACC94012707 on Jan. 27, 1994, and MVA 575, deposited under ECACC 00120707on Dec. 7, 2000. MVA-BN®, deposited on Aug. 30, 2000 at the EuropeanCollection of Cell Cultures (ECACC) under number V00083008, and itsderivatives, are additional exemplary strains.

Although MVA-BN® is preferred for its higher safety (less replicationcompetent), all MVAs are suitable for this invention. According to anembodiment of the present invention, the MVA strain is MVA-BN® and itsderivatives. See PCT/EP01/13628, which is hereby incorporated byreference.

In certain embodiments, a recombinant MVA expresses a tumor-associatedantigen. In one embodiment, tumor-associated antigen is PSA. In oneembodiment, tumor-associated antigen is PAP. In a preferred embodiment,The MVA expresses two tumor-associated antigens, preferably a PSA and aPAP antigen. In one embodiment, the two tumor-associated antigenscomprise the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2. In oneembodiment, the two tumor-associated antigens are expressed from acassette comprising the nucleotide sequence of SEQ ID NO:3.

In further embodiments, the tumor-associated antigen is modified toinclude one or more foreign T_(H) epitopes. As described herein, suchcancer immunotherapeutic agents, are useful for the prophylaxis and/ortreatment of cancer, including cancer metastasis. The invention allowsfor the use of such agents in prime/boost vaccination regimens of humansand other mammals, including immune-compromised patients; and inducingboth humoral and cellular immune responses, such as inducing a Th1immune response in a pre-existing Th2 environment.

The term “polypeptide” refers to a polymer of two or more amino acidsjoined to each other by peptide bonds or modified peptide bonds. Theamino acids may be naturally occurring as well as non-naturallyoccurring, or a chemical analogue of a naturally occurring amino acid.The term also refers to proteins, i.e. functional biomoleculescomprising at least one polypeptide; when comprising at least twopolypeptides, these may form complexes, be covalently linked, or may benon-covalently linked. The polypeptide(s) in a protein can beglycosylated and/or lipidated and/or comprise prosthetic groups.

The term “not capable of reproductive replication” in human cell linessuch as the cell lines HaCAT (Boukamp et al. 1988, J Cell Biol 106(3):761-71) or HeLa is used in the present application as defined in WO02/42480. Thus, a virus that is “not capable of reproductivereplication” in a cell line is a virus that shows an amplification ratioof less than 1 in the cell line. The “amplification ratio” of a virus isthe ratio of virus produced from an infected cell (Output) to the amountoriginally used to infect the cells in the first place (Input). A ratioof “1” between Output and Input defines an amplification status whereinthe amount of virus produced from the infected cells is the same as theamount initially used to infect the cells. According to an embodiment ofthe present invention the viruses that are “not capable of reproductivereplication” in human cell lines may have an amplification ratio of 1.0(average value) or less, or even 0.8 (average value) or less, in any ofthe above human cell lines HeLa, HaCat and 143B.

In certain embodiments, the MVA is MVA-BN®, deposited on Aug. 30, 2000,at the European Collection of Cell Cultures (ECACC) under numberV00083008, and described in U.S. Pat. Nos. 6,761,893 and 6,193,752. Asdescribed in those patent publications, MVA-BN® does not reproductivelyreplicate in cell lines 293, 143B, HeLa and HaCat. In particular,MVA-BN® exhibits an amplification ratio of 0.05 to 0.2 in the humanembryo kidney cell line 293. In the human bone osteosarcoma cell line143B, MVA-BN® exhibits an amplification ratio of 0.0 to 0.6. MVA-BN®exhibits an amplification ratio of 0.04 to 0.8 in the human cervixadenocarcinoma cell line HeLa, and 0.02 to 0.8 in the human keratinocytecell line HaCat. MVA-BN® has an amplification ratio of 0.01 to 0.06 inAfrican green monkey kidney cells (CV1: ATCC No. CCL-70).

The amplification ratio of MVA-BN® is above 1 in chicken embryofibroblasts (CEF: primary cultures) as described in U.S. Pat. Nos.6,761,893 and 6,193,752. The virus can be easily propagated andamplified in CEF primary cultures with a ratio above 500.

In certain embodiments, a recombinant MVA is a derivative of MVA-BN®.Such “derivatives” include viruses exhibiting essentially the samereplication characteristics as the deposited strain (ECACC No.V00083008), but exhibiting differences in one or more parts of itsgenome. The “derivatives” need not be derived from MVA-BN®. Viruseshaving the same “replication characteristics” as the deposited virus areviruses that replicate with similar amplification ratios as thedeposited strain in CEF cells and the cell lines, HeLa, HaCat and 143B;and that show similar replication characteristics in vivo, asdetermined, for example, in the AGR129 transgenic mouse model.

The invention encompasses MVA viruses that have one or both of thefollowing properties of MVA-BN:

-   -   capability of reproductive replication in chicken embryo        fibroblasts (CEF), but no capability of reproductive replication        in the human keratinocyte cell line (HaCaT), the human embryo        kidney cell line (293), the human bone osteosarcoma cell line        (143B), and the human cervix adenocarcinoma cell line (HeLa);        and    -   failure to replicate in a mouse model that is capable of        producing mature B and T cells and as such is severely immune        compromised and highly susceptible to a replicating virus.

In certain embodiments, the MVA is a recombinant vaccinia virus thatcontains additional nucleotide sequences that are heterologous to thevaccinia virus. In certain such embodiments, the heterologous sequencescode for epitopes that induce a response by the immune system. Thus, incertain embodiments, the recombinant MVA is used to vaccinate againstthe proteins or agents comprising the epitope. In a preferredembodiment, the epitope is a tumor-associated antigen, preferably, PSAor PAP. In one embodiment, the PSA antigen comprises the sequence of SEQID NO:1. In one embodiment, the PAP antigen comprises the sequence ofSEQ ID NO:2.

In certain embodiments, a heterologous nucleic acid sequence is insertedinto a non-essential region of the virus genome. In certain of thoseembodiments, the heterologous nucleic acid sequence is inserted at anaturally occurring deletion site of the MVA genome as described inPCT/EP96/02926. Methods for inserting heterologous sequences into thepoxviral genome are known to a person skilled in the art. In certainembodiments, the heterologous nucleic acid sequence is inserted in anintergenic region of the MVA genome as described in published U.S.patent application 20050244428. In one embodiment, the intergenic regionis IGR 014L/015L.

In certain embodiments, pharmaceutical compositions comprise one or morepharmaceutically acceptable and/or approved carriers, additives,antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Suchadditives include, for example, but not limited to, water, saline,glycerol, ethanol, wetting or emulsifying agents, and pH bufferingsubstances. Exemplary carriers are typically large, slowly metabolizedmolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, lipidaggregates, or the like.

For the preparation of vaccines, the MVA can be converted into aphysiologically acceptable form. In certain embodiments, suchpreparation is based on experience in the preparation of poxvirusvaccines used for vaccination against smallpox, as described, forexample, in Stickl, H. et al., Dtsch. med. Wschr. 99:2386-2392 (1974).

An exemplary preparation follows. Purified virus is stored at −80° C.with a titer of 5×10⁸ TCID₅₀/ml formulated in 10 mM Tris, 140 mM NaCl,pH 7.4. For the preparation of vaccine shots, e.g., 10²-10⁸ particles ofthe virus can be lyophilized in phosphate-buffered saline (PBS) in thepresence of 2% peptone and 1% human albumin in an ampoule, preferably aglass ampoule. Alternatively, the vaccine shots can be prepared bystepwise, freeze-drying of the virus in a formulation. In certainembodiments, the formulation contains additional additives such asmannitol, dextran, sugar, glycine, lactose, polyvinylpyrrolidone, orother additives, such as, including, but not limited to, antioxidants orinert gas, stabilizers or recombinant proteins (e.g. human serumalbumin) suitable for in vivo administration. The ampoule is then sealedand can be stored at a suitable temperature, for example, between 4° C.and room temperature for several months. However, as long as no needexists, the ampoule is stored preferably at temperatures below −20° C.

In various embodiments involving vaccination or therapy, thelyophilisate is dissolved in 0.1 to 0.5 ml of an aqueous solution,preferably physiological saline or Tris buffer, and administered eithersystemically or locally, i.e., by parenteral, subcutaneous, intravenous,intramuscular, intranasal, intradermal, or any other path ofadministration known to a skilled practitioner. Optimization of the modeof administration, dose, and number of administrations is within theskill and knowledge of one skilled in the art.

In certain embodiments, attenuated vaccinia virus strains are useful toinduce immune responses in immune-compromised animals, e.g., monkeys(CD4<400/μl of blood) infected with SIV, or immune-compromised humans.The term “immune-compromised” describes the status of the immune systemof an individual that exhibits only incomplete immune responses or has areduced efficiency in the defense against infectious agents.

Certain Exemplary Tumor-Associated Antigens

In certain embodiments, an immune response is produced in a subjectagainst a cell-associated polypeptide antigen. In certain suchembodiments, a cell-associated polypeptide antigen is a tumor-associatedantigen.

In certain embodiments, a cell-associated polypeptide antigen is aself-protein antigen other than a tumor-associated antigen, which isrelated to various pathological processes, or a viral antigen, orantigens derived from an intracellular parasite or bacterium. In certaininstances, such pathogen-associated antigens are often relatively poorimmunogens (e.g. antigens from mycobacteria such as Mycobacteriumtuberculosis and Mycobacterium leprae, but also from protozoans such asPlasmodium spp.).

Numerous tumor-associated antigens are known in the art. Exemplarytumor-associated antigens include, but are not limited to, 5alphareductase, alpha-fetoprotein, AM-1, APC, April, BAGE, beta-catenin,Bcl12, bcr-abl, CA-125, CASP-8/FLICE, Cathepsins, CD19, CD20, CD21,CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55, C59, CDC27,CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyltransferase, FGF8b, FGF8a, FLK-1/KDR, folic acid receptor, G250,GAGE-family, gastrin 17, gastrin-releasing hormone, GD2/GD3/GM2, GnRH,GnTV, GP1, gp100/Pmel17, gp-100-in4, gp15, gp75/TRP-1, hCG, heparanse,Her2/neu, HMTV, Hsp70, hTERT, IGFR1, IL-13R, iNOS, Ki67, KIAA0205,K-ras, H-ras, N-ras, KSA, LKLR-FUT, MAGE-family, mammaglobin, MAP17,melan-A/MART-1, mesothelin, MIC A/B, MT-MMPs, mucin, NY-ESO-1,osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, PDGF,uPA, PRAME, probasin, progenipoientin, PSA, PAP, PSM, RAGE-1, Rb, RCAS1,SART-1, SSX-family, STAT3, STn, TAG-72, TGF-alpha, TGF-beta,Thymosin-beta-15, TNF-alpha, TP1, TRP-2, tyrosinase, VEGF, ZAG, p16INK4,and glutathione-S-transferase. Particular examples of tumor-associatedantigens in prostate cancer include, but are not limited to, PSA,prostate specific membrane antigen (PSMA), PAP, and prostate stem cellantigen (PSCA).

PSA and PAP Antigens

The invention encompasses PSA and PAP antigens that are full length orfragments of PSA and PAP. Preferably, the PSA and PAP antigens arehuman. In another embodiment, the PSA and/or PAP is rat or mouse. In apreferred embodiment, the PSA and PAP antigens are encoded by thenucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively.

In one embodiment, the PAP antigen is a fragment of PAP. Preferredfragments comprise amino acids 181-95, 112-120, 133-152, 155-163,173-192, 199-213, 228-242, 248-257, 299-307, or 308-322 of human PAP.See Waeckerle-Men et al., Cancer Immunol. Immunother. 55:1524-1533(2006); Klyushnenkova et al., Prostate 67(10):1019-28 (2007); Matsuedaet al., Clin Cancer Res. 11(19 Pt 1):6933-43 (2005); Harada et al.,Oncol Rep. 12(3):601-7 (2004); Machlenkin et al., Cancer Res.65(14):6435-6442 (2005); and McNeel et al., Cancer Res. 61(13):5161-7(2001), which are hereby incorporated by reference. In one embodiment,the polypeptide comprises one of these epitopes. In other embodiments,the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 of theseepitopes. Each of the possible combinations of these epitopes isspecifically contemplated.

In certain embodiments, the fragment of PAP comprises 25, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, or 375 consecutive aminoacids of human PAP. Fragments of PAP can be assayed for the retention ofepitopes using well-known assays in the art. See, e.g., Klyushnenkova etal., Prostate 67(10):1019-28 (2007); Matsueda et al., Clin Cancer Res.11(19 Pt 1):6933-43 (2005), which are hereby incorporated by reference.

DNAs encoding these fragments can be generated by PCR or other routinemolecular biology techniques.

In one embodiment, the PSA antigen is a fragment of PSA. Preferredfragments comprise amino acids 16-24 or 154-163 of human PSA. SeeWaeckerle-Men et al., Cancer Immunol. Immunother. 55:1524-1533 (2006);Matsueda et al., Clin Cancer Res. 11(19 Pt 1):6933-43 (2005), which arehereby incorporated by reference. In one embodiment, the polypeptidecomprises one of these epitopes. In other embodiments, the polypeptidecomprises both of these epitopes.

Fragments of PSA can be assayed for the retention of epitopes usingwell-known assays in the art, such as epitope-scanning. In certainembodiment, the fragment of PSA comprises 25, 50, 75, 100, 125, 150,175, 200, 225, or 250 consecutive amino acids of human PSA.

DNAs encoding these fragments can be generated by PCR or other routinemolecular biology techniques.

Various modified PAP and PSA polypeptide antigens and methods can beproduced by methods well-known in the art. For example, the methodsdescribed in U.S. Pat. No. 7,005,498 and U.S. Patent Pub. Nos.2004/0141958 and 2006/0008465, which are hereby incorporated byreference, can be used.

The following parameters should be considered:

-   -   1. Known and predicted CTL epitopes;    -   2. Homology to related proteins;    -   3. Conservation of cysteine residues;    -   4. Predicted loop, a-helix and B-sheet structures;    -   5. Potential N-glycosylation sites;    -   6. Prediction of exposed and buried amino acid residues;    -   7. Domain organization.

Regions with a high degree of homology with other related proteins arelikely to be structurally important for the “overall” tertiarystructure, and hence for antibody recognition, whereas regions with lowhomology possibly can be exchanged with only local alterations of thestructure as the consequence.

Cysteine residues are often involved in intramolecular disulphide bridgeformation and are thus involved in the tertiary structure and should notbe changed. Regions predicted to form alpha-helix or beta-sheetstructures should be avoided as insertion points of foreign epitopes, asthese regions are thought to be involved in folding of the protein.

Potential N-glycosylation sites should be conserved if mannosylation ofthe protein is desired.

Regions predicted (by their hydrophobic properties) to be interior inthe molecule preferably should be conserved as these could be involvedin the folding. In contrast, solvent exposed regions could serve ascandidate positions for insertion of the model TH epitopes P2 and P30.

Finally, the domain organization of the protein should be taken intoconsideration because of its relevance for protein structure andfunction.

The effect of modifications of PSA and PAP can be assayed byimmunizations of animals, such as mice, to determine the effect of themodifications on humoral and cellular immune responses.

Modified Tumor-Associated Antigens

In certain embodiments, a cell-associated polypeptide antigen ismodified such that a CTL response is induced against a cell whichpresents epitopes derived from a polypeptide antigen on its surface,when presented in association with an MHC Class I molecule on thesurface of an APC. In certain such embodiments, at least one firstforeign T_(H) epitope, when presented, is associated with an MHC ClassII molecule on the surface of the APC. In certain such embodiments, acell-associated antigen is a tumor-associated antigen.

Exemplary APCs capable of presenting epitopes include dendritic cellsand macrophages. Additional exemplary APCs include any pino- orphagocytizing APC, which is capable of simultaneously presenting 1) CTLepitopes bound to MHC class I molecules and 2) T_(H) epitopes bound toMHC class II molecules.

In certain embodiments, modifications to PSA and/or to PAP are made suchthat, after administration to a subject, polyclonal antibodies areelicited that predominantly react with PSA and/or to PAP. Suchantibodies could attack and eliminate tumor cells as well as preventmetastatic cells from developing into metastases. The effector mechanismof this anti-tumor effect would be mediated via complement and antibodydependent cellular cytotoxicity. In addition, the induced antibodiescould also inhibit cancer cell growth through inhibition of growthfactor dependent oligo-dimerisation and internalisation of thereceptors. In certain embodiments, such modified PSA and/or to PAPpolypeptide antigens could induce CTL responses directed against knownand/or predicted PSA and/or to PAP epitopes displayed by the tumorcells. In a preferred embodiment, the PSA and PAP antigens induce a Bcell and a T cell response against these antigens.

In certain embodiments, a modified PSA and/or to PAP polypeptide antigencomprises a CTL epitope of the cell-associated polypeptide antigen and avariation, wherein the variation comprises at least one CTL epitope of aforeign T_(H) epitope. Certain such modified PSA and/or to PAPpolypeptide antigens comprising at least one CTL epitope and a variationcomprising at least one CTL epitope of a foreign T_(H) epitope, andmethods of producing the same, are described in U.S. Pat. No. 7,005,498and U.S. Patent Pub. Nos. 2004/0141958 and 2006/0008465.

In certain embodiments, a foreign T_(H) epitope is a naturally-occurring“promiscuous” T-cell epitope. Such promiscuous T-cell epitopes areactive in a large proportion of individuals of an animal species or ananimal population. In certain embodiments, a vaccine comprises suchpromiscuous T-cell epitopes. In certain such embodiments, use ofpromiscuous T-cell epitopes reduces the need for a very large number ofdifferent CTL epitopes in the same vaccine. Exemplary promiscuous T-cellepitopes include, but are not limited to, epitopes from tetanus toxin,including but not limited to, the P2 and P30 epitopes (Panina-Bordignonet al., 1989), diphtheria toxin, Influenza virus hemagluttinin (HA), andP. falciparum CS antigen.

Additional promiscuous T-cell epitopes include peptides capable ofbinding a large proportion of HLA-DR molecules encoded by the differentHLA-DR. See, e.g., WO 98/23635 (Frazer I H et al., assigned to TheUniversity of Queensland); Southwood S et. al, J. Immunol. 160:3363-3373(1998); Sinigaglia F et al., Nature 336:778 780 (1988); Rammensee H G etal., Immunogenetics 41(4):178-228 (1995); Chicz R M et al., J. Exp. Med178:27-47 (1993); Hammer J et al., Cell 74:197-203 (1993); and Falk K etal., Immunogenetics 39:230-242 (1994). The latter reference also dealswith HLA-DQ and -DP ligands. All epitopes listed in these references arerelevant as candidate natural epitopes as described herein, as areepitopes which share common motifs with these.

In certain other embodiments, the promiscuous T-cell epitope is anartificial T-cell epitope which is capable of binding a large proportionof haplotypes. In certain such embodiments, the artificial T-cellepitope is a pan DR epitope peptide (“PADRE”) as described in WO95/07707 and in the corresponding paper Alexander J et al., Immunity1:751-761 (1994).

Recombinant MVA

The invention encompasses a recombinant MVA virus expressing apolypeptide comprising a PAP antigen. Preferably, MVA virus expresses ahuman PAP antigen. In one embodiment, the MVA virus expresses a rat ormouse PAP antigen. In one embodiment, MVA virus encodes a full lengthPAP antigen. In a preferred embodiment, the MVA comprises the nucleotidesequence of SEQ ID NO:2.

In another embodiment, the MVA encodes a fragment of a PAP. Fragments ofPAP can be assayed for the retention of epitopes using well-known assaysin the art. See, e.g., Klyushnenkova et al., Prostate 67(10):1019-28(2007); Matsueda et al., Clin Cancer Res. 11(19 Pt 1):6933-43 (2005);Machlenkin et al., Cancer Res. 65(14):6435-6442 (2005), which are herebyincorporated by reference. In certain embodiment, the fragment of PAPcomprises 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,or 375 consecutive amino acids of human PAP.

In preferred embodiments, the MVA encodes a polypeptide comprising aminoacids 81-95, 112-120, 133-152, 155-163, 173-192, 199-213, 228-242,248-257, 299-307, or 308-322 of human PAP. See Waeckerle-Men et al.,Cancer Immunol. Immunother. 55:1524-1533 (2006); Klyushnenkova et al.,Prostate 67(10):1019-28 (2007); Matsueda et al., Clin Cancer Res. 11(19Pt 1):6933-43 (2005); Harada et al., Oncol Rep. 12(3):601-7 (2004);Machlenkin et al., Cancer Res. 65(14):6435-6442 (2005); and McNeel etal., Cancer Res. 61(13):5161-7 (2001), which are hereby incorporated byreference. In one embodiment, the polypeptide comprises one of theseepitopes. In other embodiments, the polypeptide comprises 2, 3, 4, 5, 6,7, 8, 9, or 10 of these epitopes. Each of the possible combinations ofthese epitopes is specifically contemplated.

The invention encompasses a recombinant MVA virus expressing apolypeptide comprising a PSA antigen and a recombinant MVA virusexpressing a polypeptide comprising a PSA antigen and a polypeptidecomprising a PAP antigen. Preferably, MVA virus expresses a human PSAantigen. In one embodiment, the MVA virus expresses a rat or mouse PSAantigen. In one embodiment, MVA virus encodes a full length PSA antigen.In a preferred embodiment, the MVA comprises the nucleotide sequence ofSEQ ID NO:1.

In another embodiment, the MVA encodes a fragment of a PSA. Fragments ofPSA can be assayed for the retention of epitopes using well-known assaysin the art. In certain embodiment, the fragment of PSA comprises 25, 50,75, 100, 125, 150, 175, 200, 225, or 250 consecutive amino acids ofhuman PSA.

In preferred embodiments, the MVA encodes a polypeptide comprising aminoacids 16-24 or 154-163 of human PSA. See Waeckerle-Men et al., CancerImmunol. Immunother. 55:1524-1533 (2006); Matsueda et al., Clin CancerRes. 11(19 Pt 1):6933-43 (2005), which are hereby incorporated byreference. In one embodiment, the polypeptide comprises one of theseepitopes. In other embodiments, the polypeptide comprises both of theseepitopes.

The recombinant MVA virus can be used in an immunogenic composition toinduce B-cell and T-cell immune responses against PAP and/or PSA whenadministered to a host. In a preferred embodiment, the immunogeniccomposition induces antibodies against PAP and/or PSA when administeredto a host. The immunogenic composition can contain adjuvants, diluentsand/or stabilizers. Such additives include, for example, but not limitedto, water, saline, glycerol, ethanol, wetting or emulsifying agents, andpH buffering substances.

In one embodiment, the MVA is MVA-BN®.

In a non-limiting embodiment, recombinant MVA comprising atumor-associated antigen, e.g., MVA-BN-PRO, encoding both PSA and PAPantigens is constructed as follows. The initial virus stock is generatedby recombination in cell culture using a cell type permissive forreplication, e.g., CEF cells. Cells are both inoculated with anattenuated vaccinia virus, e.g., MVA-BN®, and transfected with arecombination plasmid (e.g., pBN217) that encodes the tumor-associatedantigen, e.g., PSA or PAP, sequence and flanking regions of the virusgenome. In one non-limiting embodiment, the plasmid pBN217 containssequences which are also present in MVA-BN® (the 014L and 015L openreading frames). The PSA and PAP cDNA sequences are inserted between theMVA-BN® sequences to allow for recombination into the MVA-BN® viralgenome. In certain embodiments, the plasmid also contains a selectioncassette comprising one or more selection genes to allow for selectionof recombinant constructs in CEF cells.

Simultaneous infection and transfection of cultures allows homologousrecombination to occur between the viral genome and the recombinationplasmid. Insert-carrying virus is then isolated, characterized, andvirus stocks prepared. In certain embodiments, virus is passaged in CEFcell cultures in the absence of selection to allow for loss of theregion encoding the selection genes, e.g., gpt and Red FluorescentProtein (RFP).

Methods of Treatment

Patients with a cancer mediated by cells over-expressing atumor-associated antigens, such as PSA and/or PAP, can be treated withrecombinant MVA encoding one or more such antigens. In a preferredembodiment, the MVA is MVA-BN®. In a particularly preferred embodiment,the MVA encodes a polypeptide comprising the nucleotide sequence of SEQID NO:1 and a second polypeptide comprising the nucleotide sequence ofSEQ ID NO:2.

The cancer is preferably a prostate cancer. In an embodiment, the canceris metastatic prostate cancer. The cancer patient can be any mammal,including a mouse or rat. Preferably, the cancer patient is a primate,most preferably, a human.

The recombinant MVA encoding one or more tumor-associated antigens(e.g., PSA or PAP) can be administered either systemically or locally,i.e., by parenteral, subcutaneous, intravenous, intramuscular,intranasal, intradermal, or any other path of administration known to askilled practitioner.

In one embodiment, 10⁵-10¹⁰ TCID₅₀ of the recombinant MVA areadministered to the patient. Preferably, 10⁷-10¹⁰ TCID₅₀ of therecombinant MVA are administered to the patient. More preferably,10⁸-10¹⁰ TCID₅₀ of the recombinant MVA are administered to the patient.Most preferably, 10⁸-10⁹ or 10⁹-10¹⁰ TCID₅₀ of the recombinant MVA areadministered to the patient. Preferably, the recombinant MVA areadministered to the patient at a dose of 1×10⁸, 2×10⁸, or 4×10⁸ TCID₅₀.

The recombinant MVA can be administered once, or at multiple times. Incertain embodiments, the recombinant MVA is administered two, three,four, or five times. Preferably, the recombinant MVA is given threetimes. Most preferably, given three times at four-week intervals. Thespacing between administrations is preferably 1-4 weeks, 1-8 weeks, 1-16weeks, and 1-52 weeks. In one embodiment, the recombinant MVA isadministered at day 0 and again at days 8 and 15. In a preferredembodiment, the dosage is escalated for subsequent administrations.

In a particularly preferred embodiment, 1×10⁸, 2×10⁸, and 4×10⁸ TCID₅₀are given three times at four-week intervals. The rationale for givingmultiple doses of the recombinant MVA is based on preclinicalimmunogenicity data in mice showing that booster treatmentssignificantly increased the anti-PSA and anti-PAP immune responses.Considering the vast immunological polymorphism in humans, giving threeor more doses can ensure that every individual can reach maximal immuneresponse.

In one embodiment, anti-PSA and/or anti-PAP antibody responses. In oneembodiment, the treatment with the recombinant MVA induces anti-PSAand/or anti-PAP T-cell immune responses. In one embodiment, thetreatment with the recombinant MVA induces anti-PSA and/or anti-PAPantibody and T-cell immune responses.

In one embodiment, the treatment with the recombinant MVA induces thespreading of T cell responses to other tumor antigens.

In one embodiment, the treatment with the recombinant MVA inhibits thegrowth of PSA (+) tumors in a prophylactic and/or therapeutic setting.In one embodiment, the treatment with the recombinant MVA inhibits thegrowth of PAP (+) tumors in a in a prophylactic and/or therapeuticsetting. In one embodiment, the treatment with the recombinant MVAinhibits the growth of PSA (+) and PAP (+) tumors in a prophylacticand/or therapeutic setting.

Combination Therapy with Cytotoxic Agents

Patients with a cancer mediated by cells over-expressing atumor-associated antigens, such as PSA and/or PAP, can be treated by thecombination of a recombinant MVA encoding one or more such antigens witha taxane. Cytotoxic agents display immunomodulatory activities atsub-tumoricidal doses that could be beneficial for vaccine efficacy. Attumoricidal doses (high doses), use of these agents concurrently, priorto, or subsequent to treatment with the recombinant MVA can be superiorto either treatment alone.

In one embodiment, the taxane is docetaxel. In another embodiment, thetaxane is paclitaxel. The taxane is preferably administered at atumoricidal dose. A “tumoricidal dose” of docetaxel is at least 50mg/m². Preferably, the tumoricidal dose of docetaxel is 75-100 mg/m²,which corresponds to a dosage of approximately 25-33 mg/kg. A“tumoricidal dose” of paclitaxel is at least 90 mg/m². Preferably, thetumoricidal dose of paclitaxel is 135-175 mg/m². A “sub-tumoricidaldose” of a taxane is a dosage below the tumoricidal dosage. The taxanecan be administered by any means known to the skilled artisan, forexample, intravenously.

In one embodiment, the taxane and the MVA encoding a polypeptidecomprising a prostate tumor specific antigen are administered at thesame time.

In one embodiment, the taxane is administered prior to the recombinantMVA. In one embodiment, the recombinant MVA is administered within 6months of the taxane administration. In certain embodiments, therecombinant MVA is administered within 3 months, within 2 months, orwithin 1 month after the taxane. In one embodiment, the recombinant MVAis administered within 21 days after the taxane. In one embodiment, therecombinant MVA is administered within 14 days after the taxane. In oneembodiment, the recombinant MVA is administered within 7 days after thetaxane. Usually, the recombinant MVA is administered at least 2 daysafter treatment with the taxane.

In one embodiment, the taxane is administered after the recombinant MVA.Usually, the recombinant MVA is administered at least 1 week prior totreatment with the taxane. In one embodiment, the recombinant MVA isadministered less than 2 years prior to the taxane. In certainembodiments, the recombinant MVA is administered less than 1 year, lessthan 6 months, or less than 3 months prior to the taxane. In oneembodiment, the recombinant MVA is administered 1-26 weeks prior to thetaxane. In one embodiment, the recombinant MVA is administered 1-9 weeksprior to the taxane. In one embodiment, the recombinant MVA isadministered 1-3 weeks prior to the taxane.

In certain embodiments, the taxane is administered both prior to andafter the recombinant MVA. In other embodiments, the recombinant MVA isadministered both prior to and after the taxane. The administration ofthe recombinant MVA and the taxane can be a single administration ormultiple administrations. For example, the administrations can be 1, 2,3, 4, 5, or 6 weeks apart.

Kits

The invention encompasses kits comprising a recombinant MVA. Therecombinant MVA may be contained in a vial or container. In oneembodiment, the recombinant MVA encodes a PAP antigen. In oneembodiment, the recombinant MVA encodes a polypeptide comprising a PSAantigen. In one embodiment, the recombinant MVA encodes a polypeptidecomprising a PSA antigen and a polypeptide comprising a PAP antigen. Invarious embodiments, kits for vaccination comprising a recombinant MVAfor the first vaccination (“priming”) in a first vial or container andfor a second or third vaccination (“boosting”) in a second or third vialor container.

In one embodiment, the kit can contain a recombinant MVA andinstructions for the administration of the recombinant MVA for theprophylaxis of prostate cancer. In one embodiment, the kit can contain arecombinant MVA and instructions for the administration of therecombinant MVA for the prophylaxis of prostate cancer after an increasein one or more prostate-tumor associated markers is detected. In apreferred embodiment, the instructions can instruct that the MVA is tobe administered for the prophylaxis of prostate cancer after it isdetermined that the circulating PSA levels have increased. In oneembodiment, the instructions can instruct that the MVA is to beadministered for the prophylaxis of prostate cancer to a male patientafter the age of 30 years old. In one embodiment, the instructions caninstruct that the MVA is to be administered for the prophylaxis ofprostate cancer to a male patient after the age of 30 years old andbefore the age of 40 years old. In one embodiment, the kit can contain arecombinant MVA and instructions for the administration of therecombinant MVA for the prophylaxis of prostate cancer after the age of40.

In one embodiment, the kit can contain a recombinant MVA andinstructions for the administration of the recombinant MVA for theprophylaxis of prostate cancer metastasis. In one embodiment, the kitcan contain a recombinant MVA and instructions for the administration ofthe recombinant MVA for the prophylaxis of prostate cancer metastasisafter an increase in a prostate tumor cell associated marker isdetected. In a preferred embodiment, the instructions can instruct thatthe MVA is to be administered for the prophylaxis of prostate cancermetastasis after it is determined that the circulating PSA levels haveincreased, and despite the absence of a detectable primary tumor. In oneembodiment, the instructions can instruct that the MVA is to beadministered for the prophylaxis of prostate cancer metastasis to a malepatient after the age of 30 years old. In one embodiment, theinstructions can instruct that the MVA is to be administered for theprophylaxis of prostate cancer metastasis to a male patient after theage of 30 years old and before the age of 40 years old. In oneembodiment, the kit can contain a recombinant MVA and instructions forthe administration of the recombinant MVA for the prophylaxis ofprostate cancer metastasis after the age of 40.

In one embodiment, the kit can contain a recombinant MVA andinstructions for the administration of the recombinant MVA for thetreatment of prostate cancer. In one embodiment, the kit can contain arecombinant MVA and instructions for the administration of therecombinant MVA for the treatment of prostate cancer after an increasein one or more prostate-tumor associated markers is detected. In apreferred embodiment, the instructions can instruct that the MVA is tobe administered for the treatment of prostate cancer after it isdetermined that the circulating PSA levels have increased. In oneembodiment, the instructions can instruct that the MVA is to beadministered for the treatment of prostate cancer, to a male patientafter the age of 30 years old. In one embodiment, the instructions caninstruct that the MVA is to be administered for the treatment ofprostate cancer, to a male patient after the age of 30 years old andbefore the age of 40 years old. In one embodiment, the kit can contain arecombinant MVA and instructions for the administration of therecombinant MVA for the treatment of prostate cancer after the age of40.

In one embodiment, the kit can contain a recombinant MVA andinstructions for the administration of the recombinant MVA prior toadministration of a tumoricidal dose of a taxane. The instructions caninstruct that the MVA is to be administered at any time point between 6months and 1 week prior to taxane administration. In preferredembodiments, the instructions instruct that the MVA is to beadministered at any time point between 3 months and 1 week, six weeksand 1 week, 1 month and 1 week, 3 weeks and 1 week, and 2 weeks and 1week prior to taxane administration. In one embodiment, the instructionscan instruct that the MVA is to be administered at any time pointbetween 1 week and 0 days prior to taxane administration.

The kit can also contain a recombinant MVA and instructions for theadministration of the recombinant MVA at the same time as administrationof a tumoricidal dose of a taxane.

The kit can also contain a recombinant MVA and instructions for theadministration of the recombinant MVA after administration of atumoricidal dose of a taxane. The instructions can instruct that the MVAis to be administered at any time point between 1 day and 6 months aftertaxane administration. In preferred embodiments, the instructionsinstruct that MVA is to be administered at any time point between 2 daysand 1 week, 2 days and 2 weeks, 2 days and 3 weeks, 2 days and 1 month,2 days and 2 months, and 2 days and 3 months, and 2 days and 6 monthsafter taxane administration. In one embodiment, the instructions caninstruct that the MVA is to be administered at any time point between 0and 2 days after taxane administration.

Examples and references are given below to illustrate the presentinvention in further detail, but the scope of the present invention isnot limited by these examples. Any variations in the exemplifiedarticles which occur to the skilled artisan are intended to fall withinthe scope of the present invention. Furthermore, the specification ismost thoroughly understood in light of the cited references, all ofwhich are hereby incorporated by reference in their entireties.

EXAMPLES Example 1 Construction of MVA-BN-PRO and Analysis of ProteinExpression in Infected Cells

To develop a prostate cancer vaccine, a recombinant vaccinia virusvector, MVA-BN-PRO, which encodes the human prostate specific antigen(PSA) and the prostate acidic phosphatase (PAP), was generated. Therecombinant vaccinia virus vector MVA-BN-PRO was derived from thehighly-attenuated vaccinia virus strain MVA-BN® (Modified Vaccinia VirusAnkara-Bavarian Nordic). MVA-BN® is strongly adapted to primary chickenembryo fibroblast (CEF) cells, and does not reproductively replicate inhuman cells. In human cells, viral genes are expressed, but noinfectious virus is produced.

Origin of the Genes

The PSA gene and PAP cDNAs were transcribed (reverse transcription) fromhuman prostate total RNA purchased from Clontech (Catalog # 6410801),using routine molecular biology techniques. PSA is a prostate specificantigen produced by the prostate and is found in an increased amount inthe blood of men who have prostate cancer, benign prostatic hyperplasia,or infection or inflammation of the prostate. PSA has been identified asa target for cell-mediated immunotherapy approaches. PAP (Prostatic AcidPhosphatase) is an enzyme measured in the blood whose levels may beelevated in patients with prostate cancer which has invaded ormetastasized elsewhere. PAP is not elevated unless the tumor has spreadoutside the anatomic prostatic capsule. Therefore this prostate tumorantigen is currently investigated as a target antigen in several humanvaccine trials, some with evidence of clinical benefit.

The sequence of the resulting amplified PSA and PAP cDNAs were confirmedto match those published. That is, the PSA cDNA (e.g. among othersGenBank M26663.1 GI:618463; synonyms: kallikrein 3; KLK3; 786 bp) andthe sequence of the PAP cDNA gene (GenBank M34840.1 GI:189620; synonyms:PAP, ACP3, ACP-3; ACPP; 1161 bp) are shown below.

Human PSA cDNA sequence (99% identity to GenBank sequence M26663.1;bold: three silent nucleotide exchanges at position 48, 54 and 237,which do not change the amino acid sequence):

(SEQ ID NO: 1) ATGTGGGTCCCGGTTGTCTTCCTCACCCTGTCCGTGACGTGGATTGG C GC TGCG CCCCTCATCCTGTCTCGGATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAG T CTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCTGA

The amino acid sequence of human PSA is:

(SEQ ID NO: 3) MWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYR KWIKDTIVANP.

Human PAP cDNA sequence (100% identity to GenBank sequence M34840.1):

(SEQ ID NO: 2) ATGAGAGCTGCACCCCTCCTCCTGGCCAGGGCAGCAAGCCTTAGCCTTGGCTTCTTGTTTCTGCTTTTTTTCTGGCTAGACCGAAGTGTACTAGCCAAGGAGTTGAAGTTTGTGACTTTGGTGTTTCGGCATGGAGACCGAAGTCCCATTGACACCTTTCCCACTGACCCCATAAAGGAATCCTCATGGCCACAAGGATTTGGCCAACTCACCCAGCTGGGCATGGAGCAGCATTATGAACTTGGAGAGTATATAAGAAAGAGATATAGAAAATTCTTGAATGAGTCCTATAAACATGAACAGGTTTATATTCGAAGCACAGACGTTGACCGGACTTTGATGAGTGCTATGACAAACCTGGCAGCCCTGTTTCCCCCAGAAGGTGTCAGCATCTGGAATCCTATCCTACTCTGGCAGCCCATCCCGGTGCACACAGTTCCTCTTTCTGAAGATCAGTTGCTATACCTGCCTTTCAGGAACTGCCCTCGTTTTCAAGAACTTGAGAGTGAGACTTTGAAATCAGAGGAATTCCAGAAGAGGCTGCACCCTTATAAGGATTTTATAGCTACCTTGGGAAAACTTTCAGGATTACATGGCCAGGACCTTTTTGGAATTTGGAGTAAAGTCTACGACCCTTTATATTGTGAGAGTGTTCACAATTTCACTTTACCCTCCTGGGCCACTGAGGACACCATGACTAAGTTGAGAGAATTGTCAGAATTGTCCCTCCTGTCCCTCTATGGAATTCACAAGCAGAAAGAGAAATCTAGGCTCCAAGGGGGTGTCCTGGTCAATGAAATCCTCAATCACATGAAGAGAGCAACTCAGATACCAAGCTACAAAAAACTTATCATGTATTCTGCGCATGACACTACTGTGAGTGGCCTACAGATGGCGCTAGATGTTTACAACGGACTCCTTCCTCCCTATGCTTCTTGCCACTTGACGGAATTGTACTTTGAGAAGGGGGAGTACTTTGTGGAGATGTACTATCGGAATGAGACGCAGCACGAGCCGTATCCCCTCATGCTACCTGGCTGCAGCCCTAGCTGTCCTCTGGAGAGGTTTGCTGAGCTGGTTGGCCCTGTGATCCCTCAAGACTGGTCCACGGAGTGTATGACCACAAACAGCCATCAAGGTACTGAGGACA GTACAGATTAG

The amino acid sequence of human PAP is:

(SEQ ID NO: 4) MRAAPLLLARAASLSLGFLFLLFFWLDRSVLAKELKFVTLVFRHGDRSPIDTFPTDPIKESSWPQGFGQLTQLGMEQHYELGEYIRKRYRKFLNESYKHEQVYIRSTDVDRTLMSAMTNLAALFPPEGVSIWNPILLWQPIPVHTVPLSEDQLLYLPFRNCPRFQELESETLKSEEFQKRLHPYKDFIATLGKLSGLHGQDLFGIWSKVYDPLYCESVHNFTLPSWATEDTMTKLRELSELSLLSLYGIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMYSAHDTTVSGLQMALDVYNGLLPPYASCHLTELYFEKGEYFVEMYYRNETQHEPYPLMLPGCSPSCPLERFAELVGPVIPQDWSTECMTTNSHQGTEDSTD.Origin of the Promoter

The A-type inclusion body promoter of cowpox virus (ATI), a latepromoter (shown below), was synthetically generated in a pBluescript KS+plasmid (Stratagene), excised and inserted in front of both the PAPsequence and the PSA sequence. Consequently, the PSA and PAP proteinshould be expressed with other late genes, after DNA replication.

Sequence of the ATI Promoter:

5′-GTTTTGAATAAAATTTTTTTATAATAAATC (SEQ ID NO: 5)Construction of the PSA/PAP-MVA-BN Recombination Plasmid

For the insertion of foreign genes into the MVA-BN® genome, anintermediate recombination plasmid that targets a specific region of theMVA-BN® genome, namely a deletion site or an intergenic (non-coding)region, can be used.

The intermediate pBNX128 plasmid contains MVA DNA sequences from theregions that flank the intergenic (non-coding) region (IGR) between the014L and 015L open reading frames (ORFs). Sequences, e.g. PSA and PAPcDNA, can be inserted between these flanking sequences. Then, when bothplasmid and MVA-BN® are present in the same CEF cell, the 014L/015Lflanking sequences mediate homologous recombination, mediating insertionof the plasmid sequences into the 014L/015L intergenic region of theMVA-BN® genome (FIG. 1A-B). The presence of a selection cassette in theinserted sequences allows for positive selection of recombinant MVA-BN®viruses.

Generation of MVA-BN-PRO

Simultaneous infection and transfection of cultures allowed homologousrecombination to occur between the viral genome and the recombinationplasmid. The resulting recombinant vaccinia vector, designatedMVA-mBN106A, containing the PSA and PAP coding region and the selectioncassette was obtained after multiple plaque purifications underselective conditions. After amplification and further plaquepurification under non-selective conditions the recombinant vacciniavirus MVA-BN-PRO, devoid of the selection cassette, was isolated.

Plaque-purified virus MVA-BN-PRO lacking the selection cassette wasprepared. Such preparation involved twelve (12) passages including four(4) plaque purifications.

The presence of the promoter-PSA-promoter-PAP sequence and absence ofparental MVA-BN® virus in MVA-BN-PRO stocks was confirmed by DNAsequencing and PCR analysis, and nested PCR was used to verify theabsence of the selection cassette (the gpt and RFP genes). A simplifiedschematic of the MVA-BN-PRO genome is shown in FIG. 2.

Example 2 PSA and PAP Co-expression in Cells Treated with MVA-BN-PRO

Simultaneous expression of the two prostate-specific antigens encoded byMVA-BN-PRO, namely human PSA and human PAP, was demonstrated in cellsincubated with MVA-BN-PRO in vitro. Cultures of CT26, a chemicallyinduced colorectal carcinoma of BALB/c mice (Brattain et al., CancerResearch 40, 2142-2146 (1980)), were incubated with MVA-BN-PRO andsupernatants were analyzed for the presence of recombinant PSA and PAP.PSA was measured using an ELISA-based PSA diagnostic kit that isutilized routinely for the screening of human serum samples (Human PSAELISA Kit, Anogen, Ontario, Canada; PSA detection range: 2-80 ng/mL).PAP was measured indirectly via its enzymatic properties using acalorimetric assay for phosphate activities (acidic phosphatase assay;PAP detection range: 4-40 ng/mL). PSA and PAP were assessed in aliquotsof the same culture supernatants collected 24 hrs after addingMVA-BN-PRO at a multiplicity of infection (MOI) ranging from 1 to 100.

As shown FIG. 3, both antigens could be detected in the supernatants ofcells incubated with MVA-BN-PRO. The amount of recombinant PSA and PAPproduced in culture was dependent on the amount of MVA-BN-PRO (MOI) andthe number of cells used in the experiment. In contrast, neither PSA norphosphatase activity indicative of PAP could be detected in thesupernatants of control cultures incubated either in media alone or withmatching MOI of MVA-BN®.

The titration of PSA and PAP calculated using reference standard plotsfor each assay revealed that similar amounts of both antigens wereproduced by cells incubated with MVA-BN-PRO. Indeed, 1043 ng/mL PSA and209 ng/mL PAP were measured in culture supernatants when CT26 wereseeded at 1×10⁵ cells per well and incubated with MVA-BN-PRO at an MOIof 10 for 48 hrs. PSA and PAP sequences are inserted in the same regionof MVA-BN® genome and their expression is driven independently by an ATIpromoter located upstream of each sequence. This insert configurationappears to confer the proper environment for optimal expression of bothrecombinant antigens. Overall, these data show that MVA-BN® representsan adequate delivery vehicle for a well-balanced and concomitantexpression of multiple transgenic antigens like PSA and PAP.

Example 3 Induction of Anti-PAP and Anti-PSA Immune Response in MiceTreated with MVA-BN-PRO

The induction of anti-PSA and anti-PAP immune responses upon treatmentwith MVA-BN-PRO was evaluated in BALB/c mice. In these studies, variousdoses of MVA-BN-PRO ranging from 2×10⁶ to 5×10⁷ TCID50 were evaluated.Blood samples were collected one day prior to each treatment and twoweeks after the final treatment and humoral responses were analyzed byELISA. Splenocytes were collected after the final treatment and cellularresponses were analyzed by ELISpot.

Induction of Anti-PSA and Anti-PAP Antibody Responses

BALB/c mice (5 animals in each group) were treated subcutaneously with5×10⁷ TCID50 of MVA-BN-PRO at day 1, 15 and 29 (q2 weeks×3). Controlanimals were treated with MVA-BN® or formulation buffer (TBS). Bloodsamples were collected before treatment, at day 14, 28, and 42. Serafrom mice of each test group were then pooled and analyzed by ELISA. Theinduction of anti-PSA and anti-PAP antibody responses was evaluatedusing commercially available purified proteins (Meridian Life Sciences,Inc., Saco, Me.) as target antigens coated onto the wells of amicrotitration plate. As shown in FIGS. 4A and 4B, anti-PSA and anti-PAPantibody responses were detected in MVA-BN-PRO-treated mice. Detectionof anti-PSA antibody titers required at least two administrations andtiters increased following the third treatment with MVA-BN-PRO.Generally, the antibody response against PAP was more modest as titerswere always lower than those induced against PSA. The low antibodyresponse observed against PAP is likely due to the weak B-cell antigenicproperty of this protein.

Induction of Anti-PSA and Anti-PAP T-Cell Responses

BALB/c mice (5 animals in each group) were treated subcutaneously witheither control (TBS), 2×10⁶ or 5×10⁷ TCID50 of MVA-BN-PRO on day 1, 15,31 (q2w×3). Spleens were collected 5 days after the last immunizationand cell suspensions from each test group were pooled for analysis. Theinduction of T-cell responses was evaluated by ELISpot that measuredIFNγ production after in vitro antigen-specific restimulation. Librariesof 15-mer peptides with 11-mer overlaps (OPLs) and covering either thefull-length of PSA or PAP amino acid sequences were used separately forrestimulation. As shown in FIG. 5, antigen-specific T-cell responseswere detected in spleen cells from the MVA-BN-PRO treatment group uponrestimulation with both PAP and PSA OPLs, while a control OPL derivedfrom human HER-2 ecd sequence had no effect (FIG. 5A). No T-cellresponses were detected in mice of the MVA-BN® (data not shown) orTBS-treated groups (FIG. 5) when cells were restimulated with PSA, PAPor HER-2 OPLs. These data indicate that MVA-BN-PRO is a potent T-cellinducer since significant numbers of antigen-specific T-cell could bedetected directly in splenocytes without ex vivo expansion.

The contribution of CD4 helper and CD8 cytotoxic T-cells to the anti-PAPand PSA T-cell responses induced in mice upon treatment with MVA-BN-PROwas examined following depletion of T-cell subset populations prior toin vitro restimulation of spleen cells. As shown FIG. 6, T-cellresponses were detected in both CD4- and CD8-depleted T-cell subsetsupon restimulation with either PSA or PAP OPL.

Overall, these studies show that repeated treatment of mice withMVA-BN-PRO induces a broad antigen-specific adaptive immune response totwo TAAs that involves antibody and both CD4 and CD8 effector cellsubtypes. As expected, the antibody response was mainly directed towardPSA while PAP, a known weak B-cell immunogen, triggered only a modestantibody response. Because PSA and PAP are essentially represented ontumor cell surface as T-cell targets in the form of antigen-presentingmolecule/peptide complexes, the activation of cellular components of theimmune system is a critical requirement for MVA-BN-PRO potency. StrongCD4 and CD8 T-cell responses were induced against both TAAs in animalstreated with all MVA-BN-PRO doses tested. Therefore, MVA-BN-PRO has thepotential to mediate the elimination of tumor cells presenting PSAand/or PAP peptides on their surface.

Example 4 Anti-tumor Activity in Mice Treated with MVA-BN-PRO

The ability of MVA-BN-PRO to affect the growth of PSA-positive tumorcells in mice was evaluated in a prophylactic as well as a therapeuticcancer tumor model. The data show that MVA-BN-PRO can inhibit tumorgrowth in both settings. Also, MVA-BN-PRO was able to inhibit the growthof PAP-positive tumor cells in mice in a therapeutic cancer tumor model.

Induction of Protective Antigen-specific Adaptive Immunity UponTreatment with MVA-BN-PRO (Prophylactic Setting)

The ability of MVA-BN-PRO to prevent tumor growth was evaluated usingtransplanted E5 cells as a prostate cancer model. E5 is a subclone ofRM11, a murine prostate tumor cell line (Elzey et al., Int. J Cancer15;94(6):842-9 (2001)) obtained after transfection of RM11 withrecombinant DNA encoding the human PSA gene. In this efficacy study,mice where immunized with MVA-BN-PRO as described above, i.e., threetimes at 3-week intervals with either TBS, MVA-BN® (5×10⁷ TCID₅₀) orMVA-BN-PRO (2×10⁶, 1×10⁷ or 5×10⁷ TCID₅₀). Mice were then challengedwith tumors by injecting 1×10⁵ E5 cells intradermally six weeks afterthe last treatment. Tumor growth was observed twice weekly thereafterand the size of solid growing tumors was measured.

As shown in FIGS. 7C to 7E, the tumors in animals pretreated with alldoses of MVA-BN-PRO grew slower than the tumors of the TBS control group(FIG. 7A), and >50% of the mice remained tumor-free for all the dosestested at the end of study (Day 29). In contrast, measurable tumors weredetected in 100% of the mice in the TBS control groups as early as Day12 post tumor challenge. On that day, measurable tumors were detected inonly 20% of the mice from all MVA-BN-PRO treatment groups. Thedifference in mean tumor sizes was statistically significant between allMVA-BN-PRO treated groups and the TBS control group at all time pointsfrom Day 12 throughout the study (FIG. 7F).

Similarly to the TBS control group, measurable tumors were detected inalmost every MVA-BN®-treated mouse (90%) as early as Day 12 post tumorchallenge (FIG. 7B). However, 2 mice in the MVA-BN®-treated group (20%)were tumor-free at the end of study (Day 29; one mouse remainedtumor-free throughout the study and tumor regression occurred in theother mouse). Also, tumors in the MVA-BN®-treated group grew slower thanthe tumors of the TBS control group until Day 22 and statisticallysignificant differences in the mean tumor sizes between these two groupswere reached at two time points (Day 19, p=0.034 and Day 22, p=0.019).The delay of tumor growth in MVA-BN®-treated mice was only transientsince similar mean tumor sizes were observed in the TBS andMVA-BN®-treated mice at all other time points (FIG. 7F).

The MVA-BN-PRO-mediated anti-tumor activity described above wasconfirmed in a repeat experiment where mice were treated with 2×10⁶TCID₅₀ MVA-BN-PRO at 2-week intervals, then challenged with tumor cellstwo weeks post-treatment. The data at Day 29 post tumor implantation areshown FIG. 8, along the matching data from FIG. 7 for the same day postimplantation. Comparable delay of tumor growth was observed in micetreated with MVA-BN-PRO in both experiments. Moreover, statisticallysignificant differences in the mean tumor sizes were reached betweenMVA-BN-PRO- and TBS-treated groups as well as between MVA-BN-PRO- andMVA-BN-treated groups (p=0.03 and p=0.021, respectively). The transienteffect of MVA-BN® observed at early time points in FIG. 7 was notdetected in the repeat experiment (data not shown). These data show thattreatment of mice with MVA-BN-PRO induces an antigen-specific adaptiveimmune response and the establishment of immune memory. When mice aresubsequently challenged two to six weeks later with tumor cells, theimmune memory is recalled and inhibits the growth of the tumor cells.

Suppression of Tumors upon Treatment with MVA-BN-PRO (TherapeuticSetting)

The ability of MVA-BN-PRO to suppress established tumors was evaluatedusing transplanted E6 cells as a prostate cancer model. E6 is a subcloneof RM11, a murine prostate tumor cell line (Elzey et al., 2001) obtainedafter transfection RM11 with recombinant DNA encoding the human PSAgene. E6 is a lower producer of PSA than E5, which was used in theprophylactic setting described above. Mice were challenged with tumorsby injecting 1×10⁵ E6 cells intradermally and treated on the same day,then on Day 8 and 15 with either TBS, MVA-BN® or MVA-BN-PRO (5×10⁶ or5×10⁷ TCID₅₀). Tumor growth was observed twice weekly thereafter and thesize of solid tumors under the skin was measured.

As shown in FIG. 9, the tumors in animals treated with MVA-BN-PRO (FIGS.9C and 9D) grew significantly slower than tumors in MVA-BN®-(FIGS. 9Aand 9B) or TBS-treated animals (FIG. 9E). In both, MVA-BN-PRO treatmentgroups, tumor size stabilization or regression was observed in 50% ofthe animals. FIG. 9F, shows the difference in mean tumor sizes betweengroups. The average tumor volume was statistically significantlydifferent between animals treated with 5×10⁶ TCID₅₀ MVA-BN-PRO and TBS-or MVA-BN®-treated control groups (p=0.014 and p=0.032, respectively)whereas statistical significance was not reached between the 5×10⁷TCID₅₀ MVA-BN-PRO-treated group and TBS control group (p=0.07). Thesedata show that treatment of mice with MVA-BN-PRO inhibits the growth ofprostate tumors in mice in the therapeutic setting.

The ability of MVA-BN-PRO to also suppress established PAP-expressingtumors was evaluated in an experimental lung metastasis model using CT26cells stably expressing human PAP. CT26 is a chemically inducedcolorectal carcinoma of BALB/c mice (Brattain et al., 1980). In thismodel, CT26-PAP cells are injected intravenously into BALB/c mice andtumor burden is assessed in the lungs where tumor nodules grow. Micewere challenged with CT26-PAP (5×10⁵) cells injected intravenously onDay 1 and treated intraperiotenally on Day 4 with a single injection ofTBS, MVA-BN (5×10⁷ TCID₅₀) or MVA-BN-PRO (2×10⁶ and 5×10⁷ TCID₅₀). Micewere then sacrificed on Day 14 and their lungs were weighed. As shown inFIG. 10, the tumor burden in mice treated with 5×10⁷ TCID₅₀ MVA-BN-PROwas significantly lower than in control mice (p<0.024); This anti-tumoractivity was dose-dependent since the lower dose of MVA-BN-PRO had noeffect. Furthermore, this anti-tumor activity was most likely mediatedby PAP-specific immune response as tumor burden in mice of the controland MVA-BN treated groups was unchanged.

These data demonstrate that treatment of mice with MVA-BN-PRO inhibitsthe growth of established PAP-positive tumors in mice. Thus, both PSAand PAP prostate antigens encoded by MVA-BN-PRO contribute to theinduction of protective immune response capable of suppressing growth ofeither PSA- or PAP-positive tumors.

Example 5 Immunogenicity of MVA-BN-PRO Across Haplotype Restriction

Immune responses results from the interaction of antigen-derivedepitopes with polymorphic antigen-presenting molecules on immunecompetent cells. A benefit of the two tumor antigens in MVA-BN-PRO isthat they potentially increase the number of tumor antigen-derivedepitopes that can interact with antigen-presenting molecules of varioushaplotypes. Consequently, it is anticipated that MVA-BN-PRO will beimmunogenic in individuals with a broader range of haplotypes thanvaccines containing a single antigen. This possibility was evaluated inpreclinical models using animals with different haplotypes. In thisexample, the vector described in Example 1 was modified to replace theATI promoter by an early/late synthetic promoter (Ps; Chakrabarti S,Sisler J R, and Moss B, BioTechniques 23: 1094-1097 (December 1997)).Consequently, the PSA and PAP protein should be expressed with otherearly and late genes throughout the complete MVA infectious phase.

Sequence of the Ps Promoter:

(SEQ ID NO: 6) 5′-AAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAT

Male BALB/c and C57BL/6 mice (5 animals in each group) were immunized ondays 1, 15, and 29 with 5×10⁷ TCID₅₀ of MVA-BN-PRO. Blood samples werecollected on day 42, and serial dilutions of pooled sera were analyzedfor the presence of anti-PSA or anti-PAP IgG by ELISA as described inExample 3. As shown in FIG. 11, high titers of anti-PSA antibodies weredetected in sera from BALB/c mice only. In contrast, although anti-PAPantibody titers were measured in sera from both mouse strains, higheranti-PAP antibody titers were detected in serum from C57BL/6 mice. Thisdata emphasizes the haplotype relationship of the immune response forspecific antigens and support the idea that multiple tumor antigens inMVA-BN-PRO should provide effective immunity in a broader range ofindividuals with different haplotypes.

Example 6 Safety and Immunogenicity of MVA-BN-PRO in Humans

MVA-BN-PRO is currently under investigation for the treatment ofpatients with prostate cancer. At the time of this application, 4patients received 1 to 3 treatments with 1E8 TCID50 of MVA-BN-PRO withno reported adverse effects. MVA-BN-PRO immunogenicity in one of thesepatients was evaluated by comparing the T cell responses to PSA and PAPpre- and post-treatment. The presence of antigen-specific gammainterferon (IFN-γ) secreting T-cells in patient peripheral bloodmononuclear cells (PBMC) was determined using an ELISpot assay.Responses were determined prior to treatment (Base) and 2 weeks afterthe third subcutaneous vaccination with 10⁸ TCID₅₀ of MVA-BN-PRO (TC3).Patient PBMC (2×10⁵) in MATIS-10% media (RPMI, Click's medium, 10% HumanAB serum, 0.5M 2-β-Mercaptoethanol, and 2% Penicillin/Streptomycin) weretransferred to hydrated wells of Multiscreen 96-well PVDF plates(Millipore, Cat. No. MSIPS4510) pre-coated with an anti-human IFN-γcapture antibody (Mabtech, clone MAb 1D1K, Cat. No. 3420-3) at 10 μg/mL.Subsequently, PBMC were stimulated with either PSA at 5 μg/mL (BiodesignCat. No. A86878H), a 11-mer overlapping library of 63 15-mer peptides(OPL) derived from PSA full-length sequence at 63 μM (1 μM per peptide),PAP at 1 μg/mL (Biodesign, Cat. No. A81277H), a 11-mer OPL of 94 15-merpeptides derived from PAP full-length sequence at 94 μM (1 μM perpeptide), a pool of 44 MHC Class I peptide derived from 10 prostatecancer tumor associated antigens (TAA) at 44 μM (1 μM per peptide), apool of 15 15 MHC Class II peptides derived from 5 prostate cancer TAAat 15 μM (1 μM per peptide), or MVA (Bavarian Nordic,MVA-BN-PROD05A06-C) at a multiplicity of infection (MOI) of 10.

Sequence of the 63 peptides of PSA OPL:

MWVPVVFLTLSVTWI (a.a. 1-15 of SEQ ID NO: 3) VVFLTLSVTWIGAAP (a.a. 5-19of SEQ ID NO: 3) TLSVTWIGAAPLILS (a.a. 9-23 of SEQ ID NO: 3)TWIGAAPLILSRIVG (a.a. 13-27 of SEQ ID NO: 3) AAPLILSRIVGGWEC (a.a. 17-31of SEQ ID NO: 3) ILSRIVGGWECEKHS (a.a. 21-35 of SEQ ID NO: 3)IVGGWECEKHSQPWQ (a.a. 25-39 of SEQ ID NO: 3) WECEKHSQPWQVLVA (a.a. 29-43of SEQ ID NO: 3) KHSQPWQVLVASRGR (a.a. 33-47 of SEQ ID NO: 3)PWQVLVASRGRAVCG (a.a. 37-51 of SEQ ID NO: 3) LVASRGRAVCGGVLV (a.a. 41-55of SEQ ID NO: 3) RGRAVCGGVLVHPQW (a.a. 45-59 of SEQ ID NO: 3)VCGGVLVHPQWVLTA (a.a. 49-63 of SEQ ID NO: 3) VLVHPQWVLTAAHCI (a.a. 53-67of SEQ ID NO: 3) PQWVLTAAHCIRNKS (a.a. 57-71 of SEQ ID NO: 3)LTAAHCIRNKSVILL (a.a. 61-75 of SEQ ID NO: 3) HCIRNKSVILLGRHS (a.a. 65-79of SEQ ID NO: 3) NKSVILLGRHSLFHP (a.a. 69-83 of SEQ ID NO: 3)ILLGRHSLFHPEDTG (a.a. 73-87 of SEQ ID NO: 3) RHSLFHPEDTGQVFQ (a.a. 77-91of SEQ ID NO: 3) FHPEDTGQVFQVSHS (a.a. 81-95 of SEQ ID NO: 3)DTGQVFQVSHSFPHP (a.a. 85-99 of SEQ ID NO: 3) VFQVSHSFPHPLYDM (a.a.89-103 of SEQ ID NO: 3) SHSFPHPLYDMSLLK (a.a. 93-107 of SEQ ID NO: 3)PHPLYDMSLLKNRFL (a.a. 97-111 of SEQ ID NO: 3) YDMSLLKNRFLRPGD (a.a.101-115 of SEQ ID NO: 3) LLKNRFLRPGDDSSH (a.a. 105-119 of SEQ ID NO: 3)RFLRPGDDSSHDLML (a.a. 109-123 of SEQ ID NO: 3) PGDDSSHDLMLLRLS (a.a.113-127 of SEQ ID NO: 3) SSHDLMLLRLSEPAE (a.a. 117-131 of SEQ ID NO: 3)LMLLRLSEPAELTDA (a.a. 121-135 of SEQ ID NO: 3) RLSEPAELTDAVKVM (a.a.125-139 of SEQ ID NO: 3) PAELTDAVKVMDLPT (a.a. 129-143 of SEQ ID NO: 3)TDAVKVMDLPTQEPA (a.a. 133-147 of SEQ ID NO: 3) KVMDLPTQEPALGTT (a.a.137-151 of SEQ ID NO: 3) LPTQEPALGTTCYAS (a.a. 141-155 of SEQ ID NO: 3)EPALGTTCYASGWGS (a.a. 145-159 of SEQ ID NO: 3) GTTCYASGWGSIEPE (a.a.149-163 of SEQ ID NO: 3) YASGWGSIEPEEFLT (a.a. 153-167 of SEQ ID NO: 3)WGSIEPEEFLTPKKL (a.a. 157-171 of SEQ ID NO: 3) EPEEFLTPKKLQCVD (a.a.161-175 of SEQ ID NO: 3) FLTPKKLQCVDLHVI (a.a. 165-179 of SEQ ID NO: 3)KKLQCVDLHVISNDV (a.a. 169-183 of SEQ ID NO: 3) CVDLHVISNDVCAQV (a.a.173-187 of SEQ ID NO: 3) HVISNDVCAQVHPQK (a.a. 177-191 of SEQ ID NO: 3)NDVCAQVHPQKVTKF (a.a. 181-195 of SEQ ID NO: 3) AQVHPQKVTKFMLCA (a.a.185-199 of SEQ ID NO: 3) PQKVTKFMLCAGRWT (a.a. 189-203 of SEQ ID NO: 3)TKFMLCAGRWTGGKS (a.a. 193-207 of SEQ ID NO: 3) LCAGRWTGGKSTCSG (a.a.197-211 of SEQ ID NO: 3) RWTGGKSTCSGDSGG (a.a. 201-215 of SEQ ID NO: 3)GKSTCSGDSGGPLVC (a.a. 205-219 of SEQ ID NO: 3) CSGDSGGPLVCNGVL (a.a.209-223 of SEQ ID NO: 3) SGGPLVCNGVLQGIT (a.a. 213-227 of SEQ ID NO: 3)LVCNGVLQGITSWGS (a.a. 217-231 of SEQ ID NO: 3) GVLQGITSWGSEPCA (a.a.221-235 of SEQ ID NO: 3) GITSWGSEPCALPER (a.a. 225-239 of SEQ ID NO: 3)WGSEPCALPERPSLY (a.a. 229-243 of SEQ ID NO: 3) PCALPERPSLYTKVV (a.a.233-247 of SEQ ID NO: 3) PERPSLYTKVVHYRK (a.a. 237-251 of SEQ ID NO: 3)SLYTKVVHYRKWIKD (a.a. 241-255 of SEQ ID NO: 3) KVVHYRKWIKDTIVA (a.a.245-259 of SEQ ID NO: 3) YRKWIKDTIVANP (a.a. 249-261 of SEQ ID NO: 3)

Sequence of the 94 peptides of PAP OPL:

MRAAPLLLARAASLS (a.a. 1-15 of SEQ ID NO: 4) PLLLARAASLSLGFL (a.a. 5-19of SEQ ID NO: 4) ARAASLSLGFLFLLF (a.a. 9-23 of SEQ ID NO: 4)SLSLGFLFLLFFWLD (a.a. 13-27 of SEQ ID NO: 4) GFLFLLFFWLDRSVL (a.a. 17-31of SEQ ID NO: 4) LLFFWLDRSVLAKEL (a.a. 21-35 of SEQ ID NO: 4)WLDRSVLAKELKFVT (a.a. 25-39 of SEQ ID NO: 4) SVLAKELKFVTLVFR (a.a. 29-43of SEQ ID NO: 4) KELKFVTLVFRHGDR (a.a. 33-47 of SEQ ID NO: 4)FVTLVFRHGDRSPID (a.a. 37-51 of SEQ ID NO: 4) VFRHGDRSPIDTFPT (a.a. 41-55of SEQ ID NO: 4) GDRSPIDTFPTDPIK (a.a. 45-59 of SEQ ID NO: 4)PIDTFPTDPIKESSW (a.a. 49-63 of SEQ ID NO: 4) FPTDPIKESSWPQGF (a.a. 53-67of SEQ ID NO: 4) PIKESSWPQGFGQLT (a.a. 57-71 of SEQ ID NO: 4)SSWPQGFGQLTQLGM (a.a. 61-75 of SEQ ID NO: 4) QGFGQLTQLGMEQHY (a.a. 65-79of SEQ ID NO: 4) QLTQLGMEQHYELGE (a.a. 69-83 of SEQ ID NO: 4)LGMEQHYELGEYIRK (a.a. 74-87 of SEQ ID NO: 4) QHYELGEYIRKRYRK (a.a. 77-91of SEQ ID NO: 4) LGEYIRKRYRKFLNE (a.a. 81-95 of SEQ ID NO: 4)IRKRYRKFLNESYKH (a.a. 85-99 of SEQ ID NO: 4) YRKFLNESYKHEQVY (a.a.89-103 of SEQ ID NO: 4) LNESYKHEQVYIRST (a.a. 93-107 of SEQ ID NO: 4)YKHEQVYIRSTDVDR (a.a. 97-111 of SEQ ID NO: 4) QVYIRSTDVDRTLMS (a.a.101-115 of SEQ ID NO: 4) RSTDVDRTLMSAMTN (a.a. 105-119 of SEQ ID NO: 4)VDRTLMSAMTNLAAL (a.a. 109-123 of SEQ ID NO: 4) LMSAMTNLAALFPPE (a.a.113-127 of SEQ ID NO: 4) MTNLAALFPPEGVSI (a.a. 117-131 of SEQ ID NO: 4)AALFPPEGVSIWNPI (a.a. 121-135 of SEQ ID NO: 4) PPEGVSIWNPILLWQ (a.a.125-139 of SEQ ID NO: 4) VSIWNPILLWQPIPV (a.a. 129-143 of SEQ ID NO: 4)NPILLWQPIPVHTVP (a.a. 133-147 of SEQ ID NO: 4) LWQPIPVHTVPLSED (a.a.137-151 of SEQ ID NO: 4) IPVHTVPLSEDQLLY (a.a. 141-155 of SEQ ID NO: 4)TVPLSEDQLLYLPFR (a.a. 145-159 of SEQ ID NO: 4) SEDQLLYLPFRNCPR (a.a.149-163 of SEQ ID NO: 4) LLYLPFRNCPRFQEL (a.a. 153-167 of SEQ ID NO: 4)PFRNCPRFQELESET (a.a. 157-171 of SEQ ID NO: 4) CPRFQELESETLKSE (a.a.161-175 of SEQ ID NO: 4) QELESETLKSEEFQK (a.a. 165-179 of SEQ ID NO: 4)SETLKSEEFQKRLHP (a.a. 169-183 of SEQ ID NO: 4) KSEEFQKRLHPYKDF (a.a.173-187 of SEQ ID NO: 4) FQKRLHPYKDFIATL (a.a. 177-191 of SEQ ID NO: 4)LHPYKDFIATLGKLS (a.a. 181-195 of SEQ ID NO: 4) KDFIATLGKLSGLHG (a.a.185-199 of SEQ ID NO: 4) ATLGKLSGLHGQDLF (a.a. 189-203 of SEQ ID NO: 4)KLSGLHGQDLFGIWS (a.a. 193-207 of SEQ ID NO: 4) LHGQDLFGIWSKVYD (a.a.197-211 of SEQ ID NO: 4) DLFGIWSKVYDPLYC (a.a. 201-215 of SEQ ID NO: 4)IWSKVYDPLYCESVH (a.a. 205-219 of SEQ ID NO: 4) VYDPLYCESVHNFTL (a.a.209-223 of SEQ ID NO: 4) LYCESVHNFTLPSWA (a.a. 213-227 of SEQ ID NO: 4)SVHNFTLPSWATEDT (a.a. 217-231 of SEQ ID NO: 4) FTLPSWATEDTMTKL (a.a.221-235 of SEQ ID NO: 4) SWATEDTMTKLRELS (a.a. 225-239 of SEQ ID NO: 4)EDTMTKLRELSELSL (a.a. 229-243 of SEQ ID NO: 4) TKLRELSELSLLSLY (a.a.234-247 of SEQ ID NO: 4) ELSELSLLSLYGIHK (a.a. 237-251 of SEQ ID NO: 4)LSLLSLYGIHKQKEK (a.a. 241-255 of SEQ ID NO: 4) SLYGIHKQKEKSRLQ (a.a.245-259 of SEQ ID NO: 4) IHKQKEKSRLQGGVL (a.a. 249-263 of SEQ ID NO: 4)KEKSRLQGGVLVNEI (a.a. 253-267 of SEQ ID NO: 4) RLQGGVLVNEILNHM (a.a.257-271 of SEQ ID NO: 4) GVLVNEILNHMKRAT (a.a. 261-275 of SEQ ID NO: 4)NEILNHMKRATQIPS (a.a. 265-279 of SEQ ID NO: 4) NHMKRATQIPSYKKL (a.a.269-283 of SEQ ID NO: 4) RATQIPSYKKLIMYS (a.a. 274-287 of SEQ ID NO: 4)IPSYKKLIMYSAHDT (a.a. 277-291 of SEQ ID NO: 4) KKLIMYSAHDTTVSG (a.a.281-295 of SEQ ID NO: 4) MYSAHDTTVSGLQMA (a.a. 285-299 of SEQ ID NO: 4)HDTTVSGLQMALDVY (a.a. 289-303 of SEQ ID NO: 4) VSGLQMALDVYNGLL (a.a.293-307 of SEQ ID NO: 4) QMALDVYNGLLPPYA (a.a. 297-311 of SEQ ID NO: 4)DVYNGLLPPYASCHL (a.a. 301-315 of SEQ ID NO: 4) GLLPPYASCHLTELY (a.a.305-319 of SEQ ID NO: 4) PYASCHLTELYFEKG (a.a. 309-323 of SEQ ID NO: 4)CHLTELYFEKGEYFV (a.a. 313-327 of SEQ ID NO: 4) ELYFEKGEYFVEMYY (a.a.317-331 of SEQ ID NO: 4) EKGEYFVEMYYRNET (a.a. 321-335 of SEQ ID NO: 4)YFVEMYYRNETQHEP (a.a. 325-339 of SEQ ID NO: 4) MYYRNETQHEPYPLM (a.a.329-343 of SEQ ID NO: 4) NETQHEPYPLMLPGC (a.a. 333-347 of SEQ ID NO: 4)HEPYPLMLPGCSPSC (a.a. 337-351 of SEQ ID NO: 4) PLMLPGCSPSCPLER (a.a.341-355 of SEQ ID NO: 4) PGCSPSCPLERFAEL (a.a. 345-359 of SEQ ID NO: 4)PSCPLERFAELVGPV (a.a. 349-363 of SEQ ID NO: 4) LERFAELVGPVIPQD (a.a.353-367 of SEQ ID NO: 4) AELVGPVIPQDWSTE (a.a. 357-371 of SEQ ID NO: 4)GPVIPQDWSTECMTT (a.a. 361-375 of SEQ ID NO: 4) PQDWSTECMTTNSHQ (a.a.365-379 of SEQ ID NO: 4) STECMTTNSHQGTED (a.a. 369-383 of SEQ ID NO: 4)MTTNSHQGTEDSTD (a.a. 373-386 of SEQ ID NO: 4)

Sequence of the 44 TAA MHC Class I peptides with corresponding TAA andposition in TAA sequence:

Peptides Sequence PSMA  4-12 LLHETDSAV (a.a. 4-12 of SEQ ID NO: 7)109-117 ELAHYDVLL (a.a. 109-117 of SEQ ID NO: 7) 168-176 PSLYTKVVHY(a.a. 168-176 of SEQ ID NO: 7) 173-181 DLVYVNYAR (a.a. 173-181 of SEQ IDNO: 7) 178-186 NYARTEDFF (a.a. 178-186 of SEQ ID NO: 7) 199-207KIVIARYGK (a.a. 199-207 of SEQ ID NO: 7) 207-215 KVFRGNKVK (a.a. 207-215of SEQ ID NO: 7) 227-235 LYSDPADYF (a.a. 227-235 of SEQ ID NO: 7)260-268 NLNGAGDPL (a.a. 260-268 of SEQ ID NO: 7) 347-356 HSTNGVTRIY(a.a. 347-356 of SEQ ID NO: 7) 354-363 RIYNVIGTLR (a.a. 354-363 of SEQID NO: 7) 403-411 GTLKKEGWR (a.a. 403-411 of SEQ ID NO: 7) 431-440STEWAEENSR (a.a. 431-440 of SEQ ID NO: 7) 441-450 LLQERGVAYI (a.a.441-450 of SEQ ID NO: 7) 461-469 TLRVDCTPL (a.a. 461-469 of SEQ ID NO:7) 557-566 ETYELVEKFY (a.a. 557-566 of SEQ ID NO: 7) 641-649 EIASKFSER(a.a. 641-649 of SEQ ID NO: 7) 663-671 MMNDQLMFL (a.a. 663-671 of SEQ IDNO: 7) 680-688 GLPDRPFYR (a.a. 680-688 of SEQ ID NO: 7) 711-719ALFDIESKV (a.a. 711-719 of SEQ ID NO: 7) PSCA  7-15 ALLMAGLAL (a.a. 7-15of SEQ ID NO: 8) 14-22 ALQPGTALL (a.a. 14-22 of SEQ ID NO: 8) 21-30LLCYSCKAQV (a.a. 21-30 of SEQ ID NO: 8) 76-84 DYYVGKKNI (a.a. 76-84 ofSEQ ID NO: 8) 108-116 ALLPALGLL (a.a. 108-116 of SEQ ID NO: 8) 109-117LLPALGLLL (a.a. 109-117 of SEQ ID NO: 8) 115-123 LLLWGPGQ (a.a. 115-123of SEQ ID NO: 8) STEAP1 86-94 FLYTLLREV (a.a. 86-94 of SEQ ID NO: 9)102-116 HQQYFYKIPILVINK (a.a. 102-116 of SEQ ID NO: 9) 262-270 LLLGTIHAL(a.a. 262-270 of SEQ ID NO: 9) 292-300 MIAVFLPIV (a.a. 292-300 of SEQ IDNO: 9) PTHrp 42-51 QLLHDKGKS (a.a. 42-51 of SEQ ID NO: 10) 59-68FLHHLIAEIH (a.a. 59-68 of SEQ ID NO: 10) 59-65 FLHHLIA (a.a. 59-65 ofSEQ ID NO: 10) 165-173 TSTTSLELD (a.a. 165-173 of SEQ ID NO: 10) TARP 4-13 FPPSPLFFFL (a.a. 4-13 of SEQ ID NO: 11) 27-35 FVFLRNFSL (a.a.27-35 of SEQ ID NO: 11) 29-37 FLRNFSLML (a.a. 29-37 of SEQ ID NO: 11)Prostein 31-39 CLAAGITYV (SEQ ID NO: 12) Eph 58-66 IMNDMPIYM (SEQ ID NO:13) 550-558 VLAGVGFFI (SEQ ID NO: 14) Survivin  96-104 LTLGEFLKL (SEQ IDNO: 15) hTERT 973-981 KLFGVLRLK (SEQ ID NO: 16) HER2 665-673 VVLGVVFGI(SEQ ID NO: 17)

Sequence of the 15 TAA MHC Class II peptides with corresponding TAA andposition in TAA sequence

Kallikrein 4 125-139 SVSESDTIRSISIAS (SEQ ID NO: 18) 155-169LLANGRMPTVLQCVN (SEQ ID NO: 19) 160-174 RMPTVLQCVNVSVVS (SEQ ID NO: 20)Histone H4 14-28 GAKRHRKVLRDNIQG (a.a. 14-28 of SEQ ID NO: 21) 16-39KRHRKVLRDNIQGITKPAIRRLAR (a.a. 16-39 of SEQ ID NO: 21) 31-45TKPAIRRLARRGGVK (a.a. 31-45 of SEQ ID NO: 21) 49-63 LIYEETRGVLKVFLE(a.a. 49-63 of SEQ ID NO: 21) 71-94 TYTEHAKRKTVTAMDVVYALKRQG (a.a. 71-94of SEQ ID NO: 21) TARP  1-14 MQMFPPSPLFFFLQ (SEQ ID NO: 11) 14-27QLLKQSSRRLEHTF (SEQ ID NO: 11) ENAH (hMena) 502-510 TMNGSKSPV (SEQ IDNO: 22) PSMA 334-348 TGNFSTQKVKMHIHS (a.a. 334-348 of SEQ ID NO: 7)459-473 NYTLRVDCTPLMYSL (a.a. 459-473 of SEQ ID NO: 7) 687-701YRHVIYAPSSHNKYA (a.a. 687-701 of SEQ ID NO: 7) 730-744 RQIYVAAFTVQAAAE(a.a. 730-744 SEQ ID NO: 7)

After 40 hours of incubation at 37° C., 5% CO2, IFN-γ secretion wasdetected with 1 μg/mL of the biotinylated anti-human IFN-γ antibody(Mabtech, clone MAb 7-B6-1, Cat. No. 3420-6) followed by the addition ofStreptavidin-Alkaline Phosphatase (BD Pharmingen, Cat. No. 554065)diluted 1/5000. ELISpot plates were developed with the Vector BlueSubstrate (Vector Lab Inc., Cat. No. SK-5300) and spots were enumeratedwith an automatic spot reader (Cellular Technology Ltd. ImmunoSpot S3BAnalyzer and CTL ImmunoSpot 4.0 Professional software). As shown FIG.12, a pre-existing T cell response to PSA was detected prior toMVA-BN-PRO treatment in Patient J-D-1001. Anti-PSA T cells increasedsignificantly after treatment whereas anti-PAP T cells were detectedafter treatment only. This data indicates that MVA-BN-PRO is immunogenicin humans and that simultaneous induction of both anti-PSA and anti-PAPresponses can be achieved. MVA-BN-PRO treatment also resulted in astrong T cell response to the vector MVA-BN. Most importantly,MVA-BN-PRO treatment also resulted in the spreading of T cell responsesto other tumor antigens as illustrated by the production of IFN-γ by Tcells stimulated with TAA MHC I and II peptide pools. This indicatesthat MVA-BN-PRO-induced immune responses led to the killing of tumorcells followed by the amplification of anti-tumor responses to othertumor antigens. Antigen spreading is an important event in the inductionof anti-tumor protective immunity as it prevents tumor evasion tovaccine-induced responses. Hence, the ability of MVA-BN-PRO to mediateimmune responses to two tumor antigens in humans is a property thatprovides an effective immunotherapy.

1. A method for treating a human prostate cancer patient comprisingadministering to the human patient a recombinant modified vaccinia virusAnkara (MVA) encoding a polypeptide comprising a human prostate-specificantigen (PSA) antigen and a polypeptide comprising a human prostaticacid phosphatase (PAP) antigen.
 2. The method of claim 1, wherein theMVA is modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN).
 3. Themethod of claim 1, wherein the MVA virus comprises the nucleotidesequences of SEQ ID NO:1 and SEQ ID NO:2.
 4. The method of claim 1,wherein a nucleic acid molecule encoding the PSA antigen and the PAPantigen are inserted in the MVA intergenic region 014L/015L.
 5. Themethod of claim 1, wherein the cancer is prostate cancer in the patientis of metastasis.
 6. The method of claim 1, wherein the recombinant MVAis administered prior to a tumoricidal dose of a taxane.
 7. The methodof claim 1, wherein the recombinant MVA is administered at the same timeas a tumoricidal dose of a taxane.
 8. The method of claim 1, wherein therecombinant MVA is administered after a tumoricidal dose of a taxane. 9.The method of claim 6, wherein the taxane is docetaxel or paclitaxel.10. The method of claim 8, wherein the taxane is docetaxel orpaclitaxel.
 11. The method of claim 1 comprising administering to thepatient a priming dose of the recombinant MVA and administering one ormore boosting doses of the recombinant MVA.
 12. The method of claim 11,wherein a priming dose of 1×10⁸ TCID₅₀ and two boosting doses of 2×10⁸and 4×10⁸ TCID₅₀ are administered to the patient.
 13. The method ofclaim 12, wherein the boosting doses are given at four and eight weeksafter the priming dose.